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UNITED STATES DEPARTMENT OF AGRICULTURE 
BULLETIN No. 780 

Contribution from the Bureau of Entomology 
L. O. HOWARD, Chief 




Washington, D. C. 



PROFESSIONAL PAPER 



June 12, 1919 



NOSEMA-DISEASE 

•By 
G. F. WHITE, Specialist in Insect Diseases 



CONTENTS 



Introduction 

Name of Disease . . . . . . 

Digestive Tract of Adult Bees . . 
Cause of Nosema- Disease . . . 
A Three- Year Study of Nosema Infec 

tion in an Apiary ...... 

Symptoms of Nosema-Disease . . 
Methods Employed in Experimental 

Studies 

Effect of Nosema Infection on the Colony 

and on the Apiary 

Resistance of Nosema apis to Heating 
Resistance of Nosema apis to Drying 
Resistance of Nosema apis to Fermenta 

tion . 



Page 
1 



Page 

Resistance of Nosema apis to Putrefaction 35 

Resistance of Nosema apis to Direct 
Sunlight 

Period Nosema apis Remains Virulent . 

Infectiousness of Brood-Combs from 
Nosema-Infected Colonies .... 

Resistance of Nosema apis to Carbolic 
Acid . 

Effect of Drugs oh Nosema-Disease . . 

Modes of Transmission of Nosema-Dis- 
ease 

Diagnosis of Nosema-Disease 

Prognosis in Nosema-Disease 

Summary and Conclusions . 

Literature Cited . . .«. . . 





WASHINGTON 

GOVERNMENT PRINTING OFFICE 

1919 




, Monograph 



D« of 

JUM m 1919 



;- ■■ 



UNITED STATES DEPARTMENT OF AGRICULTURE 




BULLETIN No. 780 

Contribution from the Bureau of Entomology 
L. O. HOWARD, Chief 




Washington, D. C. 



PROFESSIONAL PAPER 



June 12, 1919 



NOSEMA-DISEASE, 

ByG. F. White, 

Specialist in Insect Diseases. 



CONTENTS. 



Page. 

Introduction 1 

Name of disease 3 

Digestive tract of adult bees 4 

Cause of Nosema-disease 7 

A three-year study of Nosema infection in an 

apiary 13 

Symptoms of Nosema-disease 21 

Methods employed in experimental studies. . 22 
Effect of Nosema infection on the colony and 

on the apiary 23 

Eesistance of Nosema apis to heating 29 

Resistance of Nosema apis to drying 31 

Resistance of Nosema apis to fermentation. . . 33 



Page. 

Resistance of Nosema apis to putrefaction ... 35 

Resistance of Nosema apis to direct sunlight . . 37 

Period Nosema apis remains virulent 39 

Infectiousness of brood-combs from Nosema- 

infected colonies , 43 

Resistance of Nosema apis to carbolic acid. . . 44 

Effect of drugs on Nosema-disease 44 

Modes of transmission of Nosema-disease 46 

Diagnosis of Nosema-disease 48 

Prognosis in Nosema-disease. 53 

Summary and conclusions 56 

Literature cited 58 



INTRODUCTION. 

Nosema-disease is an infectious disease of adult honeybees. It 
causes the death of many individual bees, tending thereby to weaken 
the colonies infected. Many colonies die of the disease, but the per- 
centage of deaths is comparatively small and entire apiaries are 
rarely, if ever, destroyed by it. It is not to be considered, therefore, 
as a particularly serious disorder. This is shown by the results 
recorded throughout the present paper. It is to be thought of rather 
as a disease the losses from which are less to the infected apiary than 
the losses from either of the foulbroods, although greater than those 

-19— Bull. 780 1 



2 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 

from sacbrood. The disease is one, however, of considerable eco- 
nomic importance. 

The fact that Nosema-disease is not a new disease deserves em- 
phasis. The knowledge of the disease and its name only are of 
recent origin. Nosema-disease, like the brood diseases, has probably 
existed among bees longer than history records the keeping of bees 
by man. Since the disease is not a new one, fear regarding additional 
losses from it would not be justified. On the other hand, as we 
know of the disorder, we may entertain the hope that the losses due 
to it may now be lessened. 

Until 1909 the existence of Nosema infection among bees was not 
generally known to beekeepers, although it had been studied some- 
what by Donhoff (1857) about a half century earlier. Zander began 
his studies a decade ago and since the appearance of his first paper 
(1909) a number of investigators have made studies on the disorder. 
In the papers which have been written concerning the infection, 
widely differing views regarding certain points have been expressed. 
To discuss these different views would be to go beyond the scope of 
the present bulletin. 

The writer began the study of Nosema infection in 1910 following 
the demonstration by him that the disorder exists in the United 
States. In pursuing these investigations the object has been not 
to devise a treatment for the disease, but rather to ascertain such 
facts concerning the disorder that the beekeepers might be able to 
devise methods for its treatment with the assurance that they would 
be not only efficient but also economical. While there is yet much to 
be learned about the disease, this object has been fairly well attained. 
Relations which the results obtained bear to practical apiculture 
should be borne in mind, therefore, in reading the paper. 

During the studies the effect of the disease on colonies and on 
apiaries, the transmission of the disease, the resistance of the infecting 
germ to heat, drying, sunlight, fermentation, putrefaction, and dis- 
infectants, and the effect of drugs on the disease are among the 
problems which have been considered. 

An earlier paper (White, 1914) refers briefly to the nature of the 
results obtained from these studies. The present bulletin gives all 
the results obtained from them which are believed to be of direct 
practical value to the beekeeper or otherwise of particular interest 
to him. The nature of the bulletin is similar, therefore, to the one 
on sacbrood (White, 1917) recently published. 1 

i As in the sacbrood paper, so in the present one, technical discussions have been purposely avoided. 
The semitechnical points which could not well be omitted are briefly explained in the sacbrood paper. 
Unless the reader is familiar with the nature of such investigations, the sacbrood bulletin will probably be 
found helpful in following the present one. 



MBMA-HHAtt. 3 

NAME OF DISEASE. 

out 60 vea: onhofJ (1857, March.) discovered small oval 

bodies upon examining microscopically the stomachs from adult bees 

h he b ipposed had died of exposure. He sent some of the bees 
to Leuckart. who after an examination of them expressed the belief 
that the oval bodies were the spores of a fungus C'Pilz"). The . - 
order was referred to by Donhoff (1857. Aiignst] by the term £; Pilz- 
sucht" (fungous " 

These observations apparently had been practically forgotten at 
the time Zander (1909) reported his studies on a disease of adult 
bees in which he found small oval bodies in the walls of stomachs 
taken from affected bees. These were in fact the parasites that 
cause the disease. To the germ Zander (1909) gave the name 

r na apis and for the disease he (1911) used the narir I isemar- 
senche 

The disorder studied by Donhoff and the one studied by Zander 
are almost without question one and the same condition. It will be 
noted that each of these men in referring to the disorder used a term 
containing a reference to the parasite considered by each, respectr~T~ ~. 
as being its cause. The term " Xosema-disease. " n which the writer 
(1914) has suggested as the common name 3 for the diseisr. is noi ■ 
new one, it will be observed, but simply an English translation of the 
term "Xosema-seuche'' used by Zander 

In Switzerland X semakrankhei: 19 nonav- iisease) (Xussbau- 
1912: Ai^s:. 1913) is the term commonly used in referring 
to the disease. In Denmark Bahr (1915) used the term "Xosema- 
sygdommen" |/ Xosema-disease). 

Tiie i:;11t "X isena-iiseise possesses lerraizL :e a vires ~'zl:'z. : :n - 
mend it : 1} It is definite, is :: in refer only 

ma apis; (2) it suggests the nature of the disease 
its ause: (3) it is readily understood: and (4) it is no 

re should be observed that Xosema-disease is nc 
dysentery. Leuckart (l v ' ' March] early raised the 
ing its relation to dysentery. The question was 

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4 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 

taken up by Brotbeck (1857). Zander (1909) in his first paper re- 
ferred to Nosema infection as a (malignant) dysentery. Other discus- 
sions have appeared from time to time in regard to such relationship 
(Maassen and Nithack, 1910; Beuhne, 1911; Maassen, 1911). 

In fact the two disorders are very different and should be con- 
sidered, for the present at least, as having no direct relation to each 
other. As both conditions are widely distributed and occur most 
frequently in the spring of the year, it is to be expected that not 
infrequently both of them may be encountered together in the same 
colony. 

Efforts have been made to determine the name by which Nosema- 
disease has been known to beekeepers in the past. In these studies 
it was found (p. 16) that the highest percentage of Nosema-infected 
bees occurred in weak colonies. Consequently in asking beekeepers 
for samples bees from weak colonies were requested. In response 
to the request made approximately 150 samples were received. 
Fully half of these contained Nosema apis. Nine representative 
beekeepers located in different sections of the country that sent 
Nosema-infected bees were asked concerning the name by which the 
colonies showing the weakened condition were known. Three replied 
spring dwindling; two, not spring dwindling; two, weak colonies; one, 
bad queen; and one, "Don't -know." None suggested paralysis and 
none dysentery. 

In reply to requests for bees from colonies showing spring dwindling 
38 samples were received from 14 beekeepers located in different 
sections of the country. Out of the 38 samples 15 upon examination 
revealed the presence of Nosema apis. From these 15 samples 314 
bees were examined, of which 70 were found to be Nosema-infected. 

Samples have been received from five beekeepers who diagnosed 
the condition in the colonies from which the bees were taken as 
paralysis. Nosema apis was not found in any of them. 

The facts indicate, it would seem, that beekeepers had not learned 
to recognize the disease produced by Nosema apis by any one name. 

DIGESTIVE TRACT OF ADULT BEES. 

In Nosema infection the parasite Nosema apis enters, infects, and 
leaves the bee by way of the digestive tract. It is well, therefore, to 
know something of the location, arrangement, appearance, and 
structure of the organs of the alimentary canal of the healthy adult 
bee in order that the disease when encountered may be recognized 
and more fully understood. 

The following description is an abbreviation of a general survey of 
the alimentary tract by Snodgrass (1910). The part of the alimentary 
canal (fig. 1) immediately following the mouth forms an enlargement 
called the pharynx (PJiy). Succeeding this is the oesophagus (CE), 



NOSEMA-DISEASE. 



a slender tube traversing the entire thorax. In the anterior part of 
the abdomen the oesophagus expands into a large thin-walled sac 
which is known as the honey stomach (HS) ; next is the short neck- 
like portion, the proventriculus (Pvent); then comes the large U- 
shaped portion, the stomach or ventriculus (Vent), an organ with 
thick walls and many annular constrictions. Following the. stomach 
is the short, narrow 
and coiled, small in- 
testine (SInt) having 
a circle of about one 
hundred long, greatly 
coiled, blind, thread- 
like tubes opening 
into its anterior end. 
These tubes are the 
Malpighian tubules 
(Mai). Following the 
small intestine is the 
large intestine or rec- 
tum (Red). When 
bees have been con- 
fined for some time 
this latter portion of 
the canal is found dis- 
tended with material 
to be voided. 

Since the stomach is 
always invaded by the 
parasite in Nosema- 
disease, and the Mal- 
pighian tubules occa- 
sionally are, a further 
description of the 
structure of these or- 
gans seems warranted. 

The stomach (fig. 
1, Vent) is a relatively 
thick-walled organ 
lying U-shaped within 
the abdomen. When 
removed and straight- 
ened it is seen to be in general cylindrical but somewhat spindle-shaped 
in form. (PI. I.) Circular constrictions present give to it a segmented 
appearance. The number and distinctness of these transverse mark- 
ings vary somewhat. The size of the organ and its color vary also. 
The color varies within wide limits, being usually some shade of 



SInr. 



Vent 




Fig. 1.— Alimentary canal of worker bee: Pharynx (Phy), 
{(E), honey stomach (HS), proventriculus (Pvent), stomach or 
ventriculus (Vent), small intestine (SInt), and large intestine or 
rectum (Red), rectal glands (RGl), Malpighian tubules (Mal) % 
Salivary glands of head (2GI) and thorax (SGI), and pharyngeal 
glands (1 Gl) are also shown. (Snodgrass.) 



6 



BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 



brown. It may be quite light, approaching a yellow, or it may be 
dark, approaching the red observed in the flesh of the ox. Stomachs 
of the lighter shades especially are translucent. 

The rather thick walls of the stomach (fig. 2) consist of an inner 
epithelial and an outer muscular portion. Between these is the 



LMd 



Pmb 




Fig. 2.— Microscopic anatomy of alimentary canal of worker bee: A , cross section of stomach showing 
peritrophic membranes {Pmb); B, wall of stomach, more highly magnified, showing epithelial layer 
(Epth), basement membrane (BM), and muscular portion; C, section of Malpighian tubule showing 
epithelium (Epih) and basement membrane (BM); D, cross section of small intestine. This portion of 
the canal, the rectum, and the oesophagus have a heavily chitinized intima. (Snodgrass.) 

basement membrane. Both . surf aces of the epithelial layer are 
irregular. This consists of epithelial cells (Epth) varying in size and 
outline. Closely associated with the outer surface of the epithelial 
layer is the basement membrane (BM). In connection with its 
inner surface is the more or less indefinite intima (Int) which possibly 



ETOSEMA-DISEASE. 7 

bears some relation to the peritrophic membranes (Pmb). Outside 
the basement membrane is the muscular portion of the stomach wall 
consisting of three (White, 1918) muscular layers (PI. II, D; and PL 
III, L). The outer and inner ones are made up of longitudinal and 
the middle one of circular fibers (fig. 3). Each layer is made up of a 
single layer of branched fibers. 

Digestion and absorption, comparable to some extent to those 
obtaining in the human stomach, are functions which have been 
attributed to the stomach of the bee. 

The Malpighian tubules (fig. 2, G MaT) empty into the alimentary 
tract at or very near the juncture of the stomach and small intestine. 
Microscopically their structure is seen to consist of a single layer of 




Fig. 3»— Longitudinal section of stomach of honeybee showing infection with Nosema apis: ep, Epithelial 
portion, containing the spores of the parasite stained black. (The younger parasites, not differentiated 
so easily by staining, are not shown; they are found toward the base of the cells reaching the basement 
membrane (BM), but do not extend beyond it. Younger spores sometimes show an unstained area at 
one end and occasionally at both ends.) m, muscular portion of stomach wall showing an outer and an 
inner longitudinal muscular layer and a middle circular one. (Author's illustration.) 

epithelial cells (fig. 2, C, Epth) and a basement membrane (B M), but no 
pronounced intima. The function attributed to these tubules is one 
comparable in a measure to that of the kidneys of the vertebrates. 

CAUSE OF NOSEMA-DISEASE. 



THE EXCITING CAUSE. 

On December 4, 1856, Donhoff (1857, August) inoculated a colony 
of bees with the oval bodies he had found in the stomachs of adult 
bees. The inoculation was made by feeding the colony the crushed 
stomachs of the infected bees in a honey suspension diluted with 
water. Upon examining stomachs from adult bees taken from the 
inoculated colony in eight days following the inoculation no spores 
were observed. In 11 days, however, they were found to be teeming 
with the parasites. A second colony was then similarly fed on Decern- 



8 BULLETIN 780, U. S. DEPARTMENT OE AGRICULTURE. 

ber 16. On the twenty-ninth of the same month all of the bees 
examined from the colony were found to be infected. The results 
of these experiments strongly indicated that the disorder in which 
the oval bodies were found was an infectious one and that the bodies 
were parasites which bore a causal relation to the disease. Other 
studies made by Donhoff (1857, September) indicated that the 
parasite was quite prevalent in Germany but that there were colonies 
apparently free from infection. 

About 50 years later Zander (1909) inoculated colonies experimen- 
tally by feeding material containing the oval bodies he had encoun- 
tered in his studies. In bees from the colonies inoculated he demon- 
strated that the oval bodies were in the walls of the stomach. This 
fact showed still more conclusively that there was an infectious 
disease of adult bees in which the oval bodies were parasites bearing 
a causal relationship to the disease. 

The oval bodies studied by Zander and those studied by Donhoff 
in all probability are the same. To Zander, however, is due the 
credit for having determined their true nature. Together with 
Doflein he (1909) classified the germ as a protozoan (a one-celled 
animal parasite) belonging to the group Microsporidia and to the 
genus Nosema. Zander gave the name Nosema apis to the species 
he found in the honeybee. 

The parasite Nosema apis grows and multiplies for the most part 
in the epithelium of the stomach (fig. 3 ; Pis. II and III) of the adult 
bee. Occasionally, but rarely, it is found within the epithelial cells 
of the Malpighian tubules (Pis. II and III). When Nosema apis is 
encountered in making an examination for the parasite it is the spore 
form (fig. 4 ; PL III, G, H) that is most often encountered and most 
readily recognized. Viewed microscopically the spore in unstained 
preparations is seen to be a small, refractile, more or less oval body 
varying somewhat in size but measuring about 2/10,000 of an inch 
in length and about 1/10,000 of an inch in width. Its width seems, 
however, to be slightly greater than one-half its length. 1 The spore 
is surrounded by a somewhat resistant coat which tends to maintain 
for it a constant form. It is not, however, a rigid structure, since, 
when studied in fresh preparations, it will be seen to bend to and fro 
as it is carried along by a current under the cover glass. 

The manner in which a bee becomes infected with Nosema apis is 
in general as follows : Spores which have left the body of an infected 
bee with the excrement are ingested by the healthy adult bee. The 
environment within the stomach of the bee is favorable for the 

1 Measurements were made of spores in smears stained with iron hematoxylin and of others in prepara- 
tions made by an India-ink method. In making the latter preparations thin smears of the spore containing 
material were made and allowed to dry, and over these smears a thin film of undiluted India ink was spread. 
The average length of the spores measured in the stained preparations was 4.15 n and the average breadth 
2.06 m; the average length in the India ink preparations was 4.46 n and the average breadth 2.44 m. 



Bui. 780, U. S. Dept. of Agriculture. 



Plate I. 




Bui. 780, U. S. Dept. of Agriculture. 



Plate 1 1 



©& 





B 




Photographs of Sections of the Stomach of the Honeybee as Seen 
Through the Miscroscope. 

A, entire cross section of stomach (queen) and Malpighian tubules, showing infection of these organs 
with Nosema apis; B, a portion of A more highly magnified; C, a small portion of a longitudinal 
section of a stomach from a healthy bee; D, similar to C, but from a Nosema-infected bee; E, infected 
epithelium highly magnified, the disease as seen in America; F, similar to E, but from a preparation 
made by Zander in Germany. (Original.) 



Bui. 780, U. S. Dept. of Agriculture. 



PLATE III. 



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K 



Further Studies on Nosema apis as Revealed by the Microscope. 

G, Nosema apis as seen in a stained smear preparation; H, a stained smear preparation showing within 
the groups how closely the cells are packed with parasites (note the nucleus of an epithelial cell below 
and to the ri?ht of the center); /, smear showing young forms (note the paired appearance); J, por- 
tions of epithelial cells are shed into the lumen of the stomach, carrying with them the contained 
parasites, accounting for the groups in this photograph; K, cross sections of Malpighian tubules 
highly magnified (the epithelial cells of the one to the left are not infected, some of those of the 
one above contain parasites, while all of those of the one to the right are heavily infected); L, tangen- 
tial section of stomach wall showing the three muscular layers, the fiber of all of them being branched 
and striated. The inner and outer layers are made up of longitudinal fibers while the middle one 
consists of circular ones. (Original.) 



NOSEMA-DISEASE. 9 

growth and multiplication of the parasite. The digestive fluids are 
believed to assist in removing the spore coat. The liberated young 
parasite finds its way to the walls of the stomach and invades the 
epithelial cells. Within this epithelial tissue it grows and multiplies 
with great rapidity, giving rise finally to numerous spores. The cells 
of the epithelium at times seem to become virtually filled with the 
parasites (fig. 3; Pis. II and III). That portion of an epithelial 
cell that is normally shed into the lumen of the stomach in case of 
infection bears with it many spores. These are liberated gradually 
from the fragments, become mixed with the partially digested food 
of the stomach, and are carried onward first into the small and then 
into the large intestine and finally pass out of the alimentary tract 
with the excrement. Other bees ingesting these spores become 
infected. This in brief is the life 
cycle ' through which the parasite 
passes. 

Nosema apis reaches the tissues of 
the bee by way of the alimentary 
tract. In infecting the stomach the 
parasite reaches the basement mem- 
brane but does not penetrate it (Pis. 
II and III). The muscular part of 
the organ is therefore uninvolved 
(fig. 3). Likewise when the infec- 
tion is found in the Malpighian 
tubules the germ does not proceed T 

i_ j t_ t_ -L ^ G * — s P ores of Nosema apisas seen in a fresh 

beyond the basement membrane preparation, indicating their general oval form. 

(Pis. II and III). Furthermore the (Original.) 

germ does not infect (fig. 1) the pharynx (Phy), the oesophagus {(E), 
the honey sac (HS), the proventriculus (Pvent), the small intestine, or 
the large intestine (Red). — organs which possess a pronounced chitin- 
ized intima. Infection with the parasite seems, therefore, to be con- 
fined to the epithelium of the stomach and of the Malpighian tubules. 
So far the writer has not encountered the germ in the blood, muscu- 
lature, or any of the other tissues of the body. 

Nosema apis has not been cultivated in pure cultures by artificial 
methods. The nature of the organism makes the accomplishment of 
such a task at the present time especially difficult. Direct proof ob- 
tained by the inoculation of bees with cultures of the parasite has 
not, therefore, been obtained. Fortunately such direct proof is not 

1 Fantham and Porter (1911 and 1912) encountered a parasite in bees taken from colonies affected with 
Isle of Wight disease which they have identified as Nosema apis. Their studies on the morphology of the 
parasite are interesting. 

The morphology of Nosema apis and of Nosema bombycis are apparently quite similar and studies made 
by Stempell (1909) on the latter parasite may be referred to with profit in studying Nosema apis. 

103789— 19— Bull. 780 2 




10 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 

always necessary to establish the causal relationship between the 
germ and the disease. 

Because of the absence of any of the higher animal parasites and 
of fungi in bees suffering from Nosema-disease these groups of para- 
sites naturally can be eliminated as possible causal factors. Maiden 
(1912, 1913) studied the bacteriology of Nosema-infected bees. He 
found that the number of bacteria in the diseased bees was much 
greater than in normal ones, the proportion being as 12 to 1. He 
found, however, no evidence of a direct etiological relation existing 
between these bacteria and the disease. Whether they play a 
secondary r61e is a question which admits of much discussion but 
one which is somewhat foreign to the present paper. 

Some preliminary experiments were made by the writer in regard 
to the possibility of the presence of a filtrable virus in Nosema- 
disease. The results obtained indicate that no such virus is present. 

By thus eliminating, at least tentatively, the higher animal para- 
sites, the fungi, the bacteria, and the filtrable viruses — groups of 
parasites which cause diseases in animals— there remains another 
group, the protozoa. Of this group there is only one species, Nosema 
apis, that is constantly present in Nosema-disease. Other protozoa 
are occasionally encountered in adult bees, but when found are 
present usually in small numbers only. The conclusion is naturally 
reached, therefore, that Nosema apis is the cause of Nosema-disease. 
Such a conclusion is in harmony with views generally accepted at the 
present time in regard to proof necessary to establish the causal re- 
lation of such a germ to the disease. 

PREDISPOSING CAUSES. 

* AGE. 

Experimental inoculations have shown that in general adult bees 
of all ages are susceptible to Nosema infection. In nature it is found 
that the youngest bees are always free from infection and that the 
old shiny bees usually are. The absence of Nosema apis in the younger 
ones may be attributed simply to the fact that they have not yet been 
infected through the taking of food containing the germ. In the case 
of the shiny bees it seems probable that they have escaped infection, 
although it is possible that some of them might have been infected 
at one time and later recovered. 

The brood does not seem to be at all susceptible to infection with 
Nosema apis. In heavily infected colonies the larvae and pupse appar- 
ently remain healthy. In these studies larvse were inoculated more 
or less directly by means of a pipette and examinations x were made 
daily following the inoculation. The spores were found mixed with 

i The examinations were made through fixing and sectioning inoculated larvae. 



NOSEMA-DISEASE. 



11 



the food within the stomach for from 1 to 3 days after the inoculation, 
but there was no evidence that the parasite had increased in numbers 
or that it had invaded the tissues. 



Nosema infection is encountered most frequently in workers, al- 
though drones and queens are susceptible. In nature it is not un- 
usual to find from 10 to 20 per cent of the workers of diseased colonies 
infected. Frequently a much higher percentage is encountered. In 
no instance has the writer found Nosema infection in drones taken 
from colonies in which the disease occurred in nature. In a few in- 
stances only were the queens that were examined from such colonies 
found to be infected. 

As a result of artificial inoculation practically 100 per cent of the 
workers of the experimental colony become infected. If drones are 
present a very large percentage of them also become infected. 

Queens in experimental colonies may or may not be found infected. 
To obtain data relative to queens a number of inoculations were 
made. Table I summarizes the experiments together with the results 
obtained. 



Table I. — Nosema infection in queens in experimental colonies. 



Date of inoculation. 



Period 




before 


Workers 


examina- 


infected. 


tion. 




Days. 


Per cent. 


8 


100 


13 


100 


16 


100 


19 


100 


22 


40 


23 


50 


48 


100 


53 


100 


23 

42 




100 


48 


100 


53 


100 


162 


100 



Results of inoculation. 



Mar. 11, 1913. 

July 12, 1913. 

Do 

Mar. 3, 1914.. 
Oct. 5, 1914.. 
Oct. 19, 1914. 
Oct. 29, 1914. 

Do. 

Feb. 4, 1915.. 
Sept. 16, 1914 
Nov. 20, 1912 
Oct. 29, 1912. 
Aug. 6, 1914. 



Queen not infected. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 
Queen Nosema infected. 

Do. 

Do. 

Do. 

Do. 



It will be seen from the foregoing table that out of the 13 experi- 
mental colonies 9 of the queens upon examination were found to be 
free from infection while the other 5 were infected. Infection in 
the queen occurs less frequently, apparently, when the inoculations 
are made in the spring and summer than when made in the autumn 
or winter. Queens in colonies inoculated and kept at room tempera- 
ture were found infected in some instances and not in others although 
practically 100 per cent of the workers in all of them became in- 
fected. 



12 BULLETIN" 780, TJ. S. DEPARTMENT OF AGRICULTURE. 

RACE. 

In experiments recorded in the present paper the bees used have 
been largely hybrids, being for the most part grade Italians. Two 
each of tested Carniolans and Caucasians and a few common blacks 
have been among the colonies used. The bees were found to be sus- 
ceptible to Nosema infection in all instances. It is not unlikely that 
future studies will show a difference among the races as to their 
relative immunity to the disease, but sufficient data are yet wanting 
to justify a definite statement in regard to the point. 



Nosema infection has been reported from Australia (Price and 
Beuhne, 1910), Brazil (Zander, 1911), Canada (White, 1914), Eng- 
land (Fantham and Porter, 1911), Germany (Zander, 1909), and 
Switzerland (Nussbaumer, 1912) . Studies have not yet been made in 
Denmark on the disease (Bahr, .1916). The writer (1914) has found 
it in samples of bees received from 27 different States of the United 
States. Out of 120 samples examined 40 contained the parasite. 
Samples showing infection were received from the coast plains and 
mountains of the East, from the plains of the Mississippi Valley, 
from the plateaus and plains of the West, and from the South and 
the North. 

The infection was found in bees received from Florida and southern 
California, but in 15 samples received from Texas it was not found. 
The data thus far obtained indicate that less infection occurs in the 
southern portion of the United States than farther north. Whether 
it is found in the Tropics or in the coldest climate in which bees are 
kept is not yet known. 

Laidlow (1911) reports that heavier infection was encountered in 
some parts of Australia than in others. Nussbaumer (1912) reported 
the infection from 14 of the cantons of Switzerland. 

The practical import of these observations in connection with the 
climate, to the beekeepers of the United States at least, is that the 
presence of the disease in a region can not be attributed entirely to 
the climatic conditions present. It is possible, however, that the 
climate of a particular region may affect somewhat the occurrence 
and the course of the disease in that locality. 

SEASON. 

Infection in apiaries has been found to occur at all seasons of the 
year, but is greatest during the spring. In the studies reported in 
the present paper (p. 20) infection was greatest in April and May, 
being greater in these months than in March. Very little of a 
definite character is known of the infection as it occurs in nature 
during the winter. Experimentally it has been found that bees are 
susceptible to infection with Nosema apis at all seasons of the year. 



NOSEMA-DISEASE. 13 

POOD. 

As is pointed out under the heading "Climate," Nosema-disease 
occurs in a wide range of localities. The food and water obtained in 
these localities naturally differ as to quality and quantity. Infection 
is found in colonies having an abundance of stores and in others 
having a scarcity. The disease is produced readily by experimental 
inoculations in colonies with much and in colonies with little stores. 
From these observations the conclusion seems to be justified that 
the r61e played by food in the causation of Nosema-disease is slight, if 
indeed it contributes at all appreciably to it. 

A THREE-YEAR STUDY OF NOSEMA INFECTION IN AN APIARY. 

The presence of Nosema infection among bees in the apiary of the 
Bureau of Entomology was discovered in May, 1910 (White, 1914) 
In April, 1912, a more or less systematic study was begun on the prev- 
alence and persistence of the infection in the apiary and was con- 
tinued until June, 1915, As the apiary was being used for other 
purposes than these studies, it was not possible to follow all of the 
colonies throughout this entire period. In Table II are summarized 
observations made during the first year of the study. 



14 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 



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16 BULLETIN 780, U. S. DEPARTMENT OE AGRICULTURE. 



From Table II it will be noted that in April there were 24 colonies 
in the apiary. Out of 240 bees examined from them during the 
month, 72 (30 per cent) were Nosema infected. The number of bees 
out of each sample of 10 was found to vary from to 10. 

During May, out of 410 bees examined 96 (23 per cent) were found 
to be Nosema infected. 1 

During June, out of 130 bees taken from 13 colonies 19 (15 per 
cent) were found to be Nosema infected. 

During July, out of 130 bees examined 21 (16 per cent) were 
found infected. 

During September, out of 170 bees examined 14 (8 per cent) proved 
to be Nosema infected. 

Out of a total of 1,140 bees examined in 1912, from April to Sep- 
tember, inclusive, 236 (20 per cent) Nosema infected bees were found. 
The number of infected bees found in the different colonies varied 
from 5 to 100 per cent. 2 

Five of the 24 colonies died. These were dead by the end of May. 
It was found that the number of infected bees present in tnem varied 
from 50 to 100 per cent. The number of infected bees in the colo- 
nies that lived varied from 5 to 33 per cent. 

All of the colonies that died were weak when first examined in the 
spring and dwindled until they disappeared. The colonies that 
lived gained in strength and behaved as healthy ones. 

The colonies that died had sufficient stores. The queen in each 
of them was apparently in good condition and brood was being 
reared. At times, indeed, the brood was in excess of the amount 
that could properly be cared for by the diminishing number of bees 
present. These and other facts which have been observed justify 
the belief that the immediate cause of death in each of the five colo- 
nies that died was the Nosema infection that was present. These 
colonies, therefore, may be said to have died of Nosema-disease. 

The number of colonies in the spring was increased during the bee 
season through swarming and by division. 

In September an experiment was begun in the apiary in which 10 
colonies were inoculated with Nosema apis. The results of these 
inoculations will be referred to later under experiment No. 1 (p. 23). 

Examinations were made in 1913 for the prevalence and persist- 
ence of Nosema infection in the apiary studied in 1912. Naturally 
the colonies present were not altogether the same as those of the 
previous year. Some of them had been lost and some represented 
the increase. The results obtained are summarized in Table III. 

1 Fractions are omitted in this paper, as a rule. 

2 As the younger bees and the older ones were avoided in selecting samples for examination, the results 
recorded in this paper show a higher percentage of Nosema-infected bees in the colonies than actually 
existed. 






NOSEMA-DISEASE. 17 

Table III. — Results obtained in 19 IS from a study of Xosema infection in an apiary. 1 



Colony No. 


Ex- 
peri- 
ment. 
No. 1. 


March. ! May. 


June. July. 


Ex- 
peri- 
ment. 


August. 


Sep- 

tem- October. 

ber. 


Per 
cent. 




12. 


25. 29. : 3. 


18. 14. 16. 


19. 


22. 


No. 2. 


9. 


25. 


23. 13. 


18. 28. 




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1 "Where the number of bees examined is small, the rate indicating the percentage frequently is not given. 



Colonies exam- 
Colonies repre- 
senting the increase in the spring are designated by the letters "&" to "o," inclusive. Colonies in experi- 
ment No. 1 are indicated by numbers; colonies in experiment No. 2, by capital letters. 



Explanation for Table III.— The method of recording results is the same as in Table IT. 
ined m 1913 that were examined in 1912 bear the same numbers in Table III as in Table II. 



From Table III it will be observed that in March, 1913, out of 270 
bees examined from the 25 colonies then in the apiary 28 (10 per cent) 
were found to be Nosema infected. 

During June bees were examined from 21 colonies, and out of 220 
bees 8 (4 per cent) were found to be infected. 

During July 21 colonies were examined and out of 260 bees 23 (9 
per cent) were found to be infected. 

During August bees from 18 colonies were examined and out of 240 
bees 11 (5 per cent) were found to be infected. 

During September, out of 170 bees from 17 colonies 43 (25 per 
cent) were found to be infected. 

During October bees were examined from 6 colonies only, and out of 
60 bees 1 (2 per cent) was found to be infected. 

Out of a total of 1,270 bees examined during the year 1913, 121 
(10 per cent) were infected, being less than the percentage found in 
1912, which was 20 per cent. The spring infection was very much 
less in 1913 than in 1912. 

103789°— 19— Bull. 780 3 



18 



BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 



The percentage of infected bees found during the spring and sum- 
mer remained quite constant, increasing unexpectedly in September. 
The reason for the increase can not be assigned at present. 

Out of the 25 colonies in the apiary in March, 1913, 1 (No. 12) died. 
As this colony contained a high percentage of Nosema-infected bees, 
and as it dwindled until it disappeared, it may be assumed that 
Nosema-disease was the immediate cause of its death. As in the 
preceding year all of the colonies that lived behaved much as do 
uninfected ones. 

In this year another experiment was begun in the apiary. This 
one is described as experiment No. 2 (p. 25). 

Studies similar to these made in 1912 and 1913 were continued 
throughout 1914 and until June, 1915. While in the main the col- 
onies of the apiary were those of the previous years, naturally there 
had been some changes. The results obtained are summarized in 
Table IV. 



Table IV. — Results obtained from May, 1914, to June, 1915, from a study of Nosema 

infection in an apiary. 





1914 


1915 


Colony No. 


May. 


June. 


July. 


Sep- 
tem- 
ber, 


No- 
vem- 
ber. 




March. 


April. 


May. 




2 


8 


12 


15 


27 


5 


8 


18 


15 


10 


23 


5 


2 


8 


25 


7 


26 


1 


0« 
2" 
1« 
1« 
0« 
0« 
1« 
0" 

1 

Op 
1« 

2« 
1" 
2« 
2« 
0" 
0« 
1» 
1* 
0« 
0« 
3« 
4» 
2« 
0« 
7« 
!• 


1p 
2p 
1« 
1p 
1p 
Op 
2p 
Op 
Op 
2« 
1p 
2« 
1p 
2p 
4p 
1p 
Op 
1p 
3p 
2p 
2p 


"2p* 


Op 
1p 
3p 
1p 
1p 
1p 
Op 

Op' 
2« 
Op 
2« 
2p 
2p 
1p 
1p 
1p 
2p 
Op 
2p 
2p 
2p 
3p 
Op 
Op 
4p 
1p 


1p 

Ik 
2» 
2h 
2h 
2* 
2h 

lh 

4 h 

lh 
lh 
0« 
2h 
lh 

4h 
3h 
2h 

lh 
2h 
3h 

lh 

3»> 
2h 


Op 
Op 
1p 
Op 
Oh 
0p 
lh 
0* 
Oh 
Op 

Oh 

U 

2h 
Oh 
Oh 
lh 
2h 
lh 
lh 
2h 

0p 

Oh 
IP 


5p 

*ip" 

2p 

ip" 
1p 
3p 
2p 
3p 

lh 

2p 
Ip 
3p 
3p 

2h 

Op 
Ip 
2p 


Op 
Op 
Ip 
Op 
Ip 
Op 
Ip 
Op 
Ip 
Op 
Op 
Op 
Op 
Op 
Op 
Ip 
Op 
Op 
Op 
Ip 
Ip 
Ip 
Op 
Ip 
Op 
3p 
Ip 


Op 
Op 
Op 

"'6p' 
Op 
Op 
Op 
Op 
Op 
Op 
Op 
Op 
Ip 
Ip 
Op 
Op 
Op 


Op 
Op 
Op 
Op 
Op 
Op 
Op 
Op 
Op 
Op 
Op 
Op 


Op 
0« 

Oe 

0« 
0« 


Oe 

0« 




0« 


0« 


Op 


Op 


Oh 


2 




3 














4 


0« 














5 




0« 


0« 


Op 


Ip 


Oh 


6 




7 


0« 
0» 


0* 
0« 














8 




5« 


0« 


D 






g 






10 


















11 


















14 


















15 


















16 












Oe 
Oe 
5« 
Oe 
0« 
0« 








17... 


Op 








1« 

0« 


b' 

Op 
Op 
Ip 


Ip 


Oh 


18 










19 










Oh 

Op 
Op 


Oh 


20 












Oh 


21 












Oh 


22 
















«7» 


Op 
Op 
Op 




0« 




S 
t 
U 

V 

w 

X 

y 

z 




0« 
Oe 
6« 
Oe 
Oe 
0« 
0« 
Oe 


0« 

Op 

2e 

0" 






"36" 






"50" 


4p 
2p 
2p 
5« 
3p 





Op 






Ip 

Oe 


Oe 


"66".. 






Oe 


"68" 


3h 

6h 


0p 

IP 

Oh 


Op 
Op 
Op 










"73" 














"82" . 








9e 
le 


9p 
3p 


le 










0« 



Explanation of Table IK— The colonies reported in Table TV for 1914 do not bear the same numbers that 
were assigned to them for 1913 in Table III except those designated by numbers in quotation marks. The 
first 9 colonies reported in the table for 1 1915 bear the same numbers they did in 1914. The identity of col- 
onies numbered by letters "s" to '%" inclusive, had been lost through changes made in the apiary. 



NOSEMA-DISEASE. 19 

Table IV shows that out of 1,050 bees examined during May, 
1914, 166 (16 per cent) were Nosema infected. 

In June, out of 700 bees examined 60 (9 per cent) were found 
infected. 

In July, out of 240 bees examined 2 (1 per cent) were infected. 

In September, 220 bees were examined and no Nosema-infected 
one was found. 

In November, 60 bees were examined and none was found 
infected. 

Out of 2,270 bees examined during the summer of 1914, 218 (10 
per cent) were found infected. 

It will be noted that during the early months of the active bee 
season of. 1914 there was a higher percentage of Nosema-infected 
bees in the apiary than during a similar period of 1913. 

Two colonies were so weak in May that they were disposed of. 
In one of these at least (No. 13) the weakness was most probably 
due to Nosema infection. 

During the first week in July the apiary was moved to a new loca- 
tion. It is interesting to note that the amount of Nosema infection 
after removal was reduced to practically nothing. This is not defi- 
nitely accounted for by the results obtained by these investigations. 1 

Examinations were made of a portion of the apiary in 1915. In 
March, out of 50 bees taken from 5 colonies, 6 (12 per cent) were 
found to be Nosema infected. 

In April, out of 280 bees taken from 17 colonies 24 (9 per cent) 
were found infected. 

In May, out of 200 bees taken from 10 colonies 16 (8 per cent) 
were infected. 

Out of 530 bees examined from the apiary during the spring of 
1915, 46 (9 per cent) infected ones were found. 

Among the colonies that were examined during the spring of 
1915 two (Nos. 8 and 18) died by the end of April. Both of these 
contained a rather high percentage of Nosema-infected bees. Two 
others containing an equal or greater number of infected bees lived 
throughout May and had recovered apparently by June. In case 
of these 4 colonies it can properly be said that the two colonies that 
died died of Nosema disease, whereas the two that lived recovered 
from it. 

In Table V is given a summary of the results obtained in the study 
of the apiary from April, 1912, to June, 1915. 

1 That the immediate environment of the apiary determines, to some extent, the presence or absence of 
Nosema-disease and its transmission seems quite likely. In searching for the cause for such a difference 
the water supply of the bees, if near by, must not be overlooked (p. 46). In this connection, it may be 
pointed out that in the experimental apiary (PI. IV) Nosema infection at no time exceeded 1 per cent, 
excepting naturally in inoculated colonies, although the source from which these colonies were obtained 
had been largely the apiary which, it will be seen from Tables II and III, showed Nosema infection in from 
10 to 20 per cent of the bees. Here there was no slowly moving body of water used by the bees as the source 
of their water supply. 



20 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 

Table V. — Summary of results from a study of Nosema infection in an apiary. 





March. 


April. 


May. 


June. 


July. 






•d 






-d 






"d 






TJ 






*d 




Year. 


1 


o 




•a 


"8 




1 


1 




T3 

§ 






1 


SI 






1 
1 


s 


8 


03 
X 
S 


1 
I 


1 


1 

e 


i 

i 


"3 

i 

Ul 




1 
03 

a 


1 


1 

H 

o 


a 

03 

a 


I 






8 


I- 




8 




I 


8 


8 


8 


8 


8 




W 


'A 


Ah 


5 


« 


ft 


« 


fc 


Ah 


M 


£ 


A< 


« 


£ 


Ah 


1912 








240 


72 


30 


410 


96 


23 


130 


19 


If! 


130 


?i 


16 


1913 


270 


28 


10 








50 


7 




220 


8 


4 


260 


23 


9 


1914 














1,050 


166 


16 


700 


60 


9 


240 


2 


1 


1915 


50 


6 


12 


280 


24 


9 


200 


16 


8 




























Total 


320 


34 


11 


520 


96 


18 


1,910 


285 


17 


1,050 


87 


8 


630 


46 


7 










Yugust. 


September. 


October. 




•d 


















• 1 


I 




























•a 


i 




T3 


8 




•d 


i 






3 




Year. 


8 






§ 


o 




§ 


a 






a 






1 

B 


■a 

i 

© 
8 


i 


1 

OS 

X 


.1 

03 


■s 


«3 
X 


2 

03 


ta 


,o 


1 


a 




8 


i 


9 

1 


a 

i 


8 




a 

03 

8 


1 


3 
o 


"o3 
O 






ffl 


fc 


Ah 


n 


g 


Ah 


PQ 


fc 


Ah 


&H 


H 


Ah 


1912 


60 
240 


14 
11 




170 
170 
220 


14 
43 


8 
25 








1,140 

1,270 

2,210 

530 


236 

228 

121 

46 


20 


1913 


5 


60 


1 




10 


1914 


10 


1915 , 


















9 
























Total 


300 




8 


560 


57 


in 


fin 


1 




5,150 


631 


1? 































From Table V it will be observed that the number of infected 
bees found at different periods of the year varied considerably. 
April and May furnished the highest percentage, being 18 and 17 
per cent respectively. In March, June, July, August, and Sep- 
tember the number of Nosema-infected bees among those examined 
was 11, 8, 7, 5, arid 10 per cent respectively. Out of 5,150 bees 
taken from the apiary from April, 1912, to June, 1915, and exam- 
ined, 631 (12 per cent) were Nosema infected. 1 

Laidlow (1911) reports that out of somewhat more than 1,500 
bees received from various parts of Australia, 17 per cent were 
found to be Nosema infected. 

From an examination of the foregoing tables it is seen that Nosema 
infection was found to be present in practically every colony of the 
apiary. Had further examinations been made of the few colonies 
in which Nosema apis was not found, "one could well expect, from 
what is known of the disease, that these, too, would have revealed 
the presence of the infection. It is seen also that the infection 
persisted throughout all seasons of the year, and that it was heaviest 

* While this three-year study was being made the apiary served for other work. It is likely that the 
attending manipulations were accompanied from time to time by a certain amount of robbing. From the 
nature of the disease, however, it is not behoved that this fact affected materially the results obtained. 



Bui. 780, U. S. Dept. of Agriculture 




NOSEMA-DISEASE. 21 

in the spring. Some colonies died as a result of the disease, while 
a greater number recovered from the infection, increased in strength, 
and behaved in all respects as healthy colonies. 

The total of all the spring counts, during the period from 1912 
to 1915, inclusive, of the apiary under study, was 94 colonies. Out 
of this number at least 12 (13 per cent) died more or less directly 
as the result of Nosema disease. An equal or greater loss to the 
apiary than this colony loss probably is the aggregate loss in strength 
sustained by colonies weakened by the infection but which recover 
from the disease. 

Naturally it is particularly unfortunate from an economic point of 
view that the highest percentage of infected bees, and consequently 
the heaviest loss in strength sustained by colonies from Nosema 
infection, occurs in the spring. 

Beuhne (1916) has reported investigations made on colonies from 
his own apiary which are similar in nature to the foregoing studies. 
The results he obtained indicate that Nosema infection in Australia 
is similar to the infection as it occurs in America. 

SYMPTOMS OF NOSEMA-DISEASE. 

Nosema-disease presents only a few symptoms. In describing 
them the colony rather than individual bees should be considered as 
the unit, since it is the colony as a whole that is of primary interest 
to beekeepers. 

Weakness is a colony symptom which invariably will be manifest if a 
sufficiently large percentage of the bees of the colony are Nosema in- 
fected and if the infection persists for a sufficient period. When only a 
small percentage of the bees are infected the weakness resulting may 
never be apparent. The loss in strength may be gradual or sudden. 

The behavior of a Nosema-infected colony is similar to that of a 
healthy one. The stores are sufficient. The queen does her work 
well. As the colony dwindles the queen usually is among the last 
handful of bees. The brood in general is normal in appearance, but 
in colonies weakened by the disease not infrequently it is seemingly 
in excess of the amount that can be properly cared for by the adult 
bees present. 

In Nosema-disease the workers especially suffer from the infection. 
An infected bee manifests no outward symptoms of the disease when 
seen among the other bees of the colony and it performs functions 
similar to those performed by healthy ones. 

When the stomach of an infected bee is removed it may show 
marked changes which are characteristic of Nosema-disease. The 
organ pales as a result of infection. The brownish yellow or dark 
reddish hue of the normal stomach is gradually lost as the disease 
advances. The organ (PL I) is often increased in size, the circular 



22 



BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 



constrictions are less marked, and the transparency is diminished. 
In late stages of the disease, however, the stomach approaches the 
normal in size and the constrictions are again well marked. The 
organ is then white and opaque and the tissues are friable and easily- 
crushed. When crushed the mass presents a milky appearance. 

Upon microscopic examination Nosema apis is found in very large 
numbers in the crushed tissues. The presence of the parasite is 
almost invariably recognized by its spore form. The presence of 
Nosema-infected bees in a colony is the one constant colony symptom 
of the disease. 

METHODS EMPLOYED IN EXPERIMENTAL STUDIES. 

As Nosema apis has not been grown in the laboratory by artificial 
methods, in carrying out these investigations it has been necessary 

to inoculate a large number of 
colonies of bees. The use of a 
few bees in cages was found to 
be inadequate for experimental 
purposes. A 4 to 6 frame nu- 
cleus in a 10-frame hive body 
(fig. 5) serves well the purposes 
of an experimental colony. 
The experimental apiary (PI. 
IV) , consisting usually of about 
50 colonies, was the same one 
that was used in the sacbrood 
studies. During the bee sea- 
son the colonies were inocu- 
lated and kept in the apiary 
in the open under conditions 
similar to those occurring in 
nature. Precautions similar 
to those observed in the sac- 
brood studies were followed in 
the present studies. During the winter colonies to be inoculated 
were removed to and kept in the laboratory. The top of the hive 
body was screened and the bees given free opportunity for flight 
through a hole in the window. 

The manner of obtaining the parasite Nosema apis from diseased 
bees for use in the inoculations is described under " Diagnosis' ' 
(p. 48). The stomachs of from 5 to 10 infected bees are amply suffi- 
cient for each inoculation. After their removal from the bees they 
are crushed, suspended in sirup, and fed to a colony free or practically 
free from Nosema infection. The methods throughout are similar 




Fig. 5.— Experimental hive, having four Hoffman 
frames, a division hoard, Petri dishes as feeders, the 
entrance nearly closed with wire cloth, and the open- 
ing on the side of the hive hody occupied by the 
frames. (Original.) 



NOSEMA-DISEASE. 23 

in general to those employed and described in the sacbrood studies. 
It should be stated in addition that no watering place for the bees 
was provided at the time of these experiments and none with sluggish 
water was near by. 

The results of an experiment usually can be determined during the 
second week following the inoculation. The diagnosis is made as 
described later in the present paper (p. 48). Usually one examina- 
tion of 10 bees is sufficient for the determination of results. It is 
advisable sometimes, however, to make others. 

As a rule experimental colonies inoculated during the summer 
recover from the infection and can be used again. The period which 
must elapse, however, before they can be used for a second experiment 
varies. An examination of the field bees should show no infection 
among them or only an occasional infected bee before another inocu- 
lation is made. A colony used in the laboratory is good for one 
inoculation only if by it Nosema-disease is produced. Should the 
results be negative following an inoculation, however, the colony may 
be used in a subsequent experiment. 

It is not necessary to disinfect a hive which has housed a Nosema- 
infected colony. The experimental colony may or may not have a 
queen. If one is present no concern need be felt in regard to whether 
or not she is infected. No fear need be entertained that drones from 
infected colonies in the apiary will transmit the infection to the 
experimental colony. 

EFFECT OF NOSEMA INFECTION ON THE COLONY AND ON THE APIARY. 

To determine the effect which Nosema infection in a colony pro- 
duces on the colony, and on the apiary of which the colony is a part, 
is a problem in the study of Nosema disease which is of vital interest 
to the beekeeper. Some observations have been made bearing 
directly upon this point. 

EXPERIMENT NO. 1. 

On September 13, 14, 15, and 18 ten colonies were fed a sirup 
suspension of the crushed intestines of Nosema-infected bees. These 
colonies (Table II, Nos. 6a, 12, 25, 35, 41, 55, 65, 66, 67, and 70) were 
in the apiary mentioned on page 13. Those selected for inoculation 
were not especially strong, the bees being easily accommodated on 
six or seven brood frames and being about an average for the apiary. 
Examinations show that about 10 per cent of the pollen-carrying 
bees of these colonies were Nosema infected at the time of the 
inoculation. The 32 uninoculated colonies in the apiary served as 
checks. 

It will be seen from Table II that after inoculation 50 to 100 per 
cent of the pollen-carrying bees in the inoculated colonies were 
Nosema infected. Out of the 100 bees examined from these colonies 



24 BULLETIN 780, U. S. DEPARTMENT OP AGRICULTURE. 

during the period from October 5 to October 16, inclusive, 132 
(70 per cent) were found infected. These colonies when examined 
on October 28 showed that, out of 100 bees examined, 78 (78 per 
cent) were infected. It will be noted, therefore, that following the 
feeding inoculations there was a marked increase in the percentage 
of Nosema-infected bees in each of the 10 colonies inoculated. 

In the experiment sufficient precautions were not taken to prevent 
robbing at the time the inoculations were made. This resulted in an 
increase also of Nosema-infected bees in some of the uninoculated 
colonies (Table II) of the apiary — the checks. The increase in the 
number of infected bees disappeared more readily from the check 
colonies, however, than from the inoculated ones, suggesting that 
probably a comparatively small amount of the contaminated sirup 
was obtained by the robbing bees. 

On December 17, out of 100 bees taken from the 10 inoculated 
colonies 49 (49 per cent) were found to be Nosema infected, showing 
that the percentage of infected bees had decreased. 

From comparison of the inoculated colonies in October and in 
December, it was observed that their strength had decreased and that 
they were relatively weaker than the checks. Toward the last of 
December one of the 10 inoculated colonies died. During the last 
week of the year the remaining 9 were packed for the winter as were 
also the check colonies. Some of the weaker check colonies were 
united, giving them a slight advantage in strength over the inoculated 
ones. 

. The winter 1912-13 being a favorable one for bees, the winter 
losses were low. In March, 1913, when the first examination of the 
apiary was made, 4 of the 10 colonies that had been inoculated had 
died out. Four of the six inoculated colonies that were still alive 
showed 4, 6, 2, and 2 Nosema-infected bees respectively in samples 
of 10 bees examined. Neither of the other two inoculated ones 
showed at the time the presence of Nosema infection. All of the 19 
uninoculated colonies packed in December were alive in March, 1913. 
Out of 190 bees caught from the entrance of these check colonies 
during March only 6 (3 per cent) were Nosema infected. 

By the middle of May another of the inoculated colonies (No. 12) 
had died, making 5 in all. Of the 10 colonies that had been inoculated 
in September, 1912, the 5 that lived through the winter and the 
following spring continued to gain in strength during the summer of 
1913 and by autumn were apparently as strong and healthy as any in 
the apiary. 

By experiment No. 1 it is shown that when colonies are inoculated 
with Nosema apis a high percentage of adult bees of each colony 
becomes Nosema infected — results which confirm similar ones 
previously obtained by Donhoff (1857), Zander (1909), and others. 



NOSEMA-DISEASE. 25 

Such results, together with facts which are recorded on the foregoing 
pages, are sufficient to demonstrate that Nosema-disease is an infec- 
tious disease of adult bees. 

It is shown also by the results of this experiment that there is a 
tendency for the infected colonies to become weakened. It is further 
shown that when inoculated in September colonies do not die out 
readily as a result of the inoculation. Furthermore the results 
indicate that the infection is not readily transmitted from the infected 
to the healthy colonies of the apiary. It is further shown that 
colonies inoculated in September may die as a result of the infection 
during the winter that follows, or they may survive the winter, gain 
in strength during the brood-rearing season, and by the following 
autumn present the appearance of healthy colonies. 

EXPERIMENT NO. 2. 

Beekeepers are always desirous of knowing whether combs from 
diseased colonies can be used in healthy ones without causing a 
spread of the infection. To obtain data relative to this point experi- 
ment No. 2 was begun in July, 1913 (Table III). In the experiment, 
brood combs from diseased colonies were inserted into colonies 
comparatively free from Nosema infection and kept under observa- 
tion for more than a year afterwards. 

Combs from the 5 colonies of experiment No. 2, which died 
during the winter and spring following their inoculation with Nosema 
apis in September, 1912, were inserted into the 6 colonies (Nos. 36, 
50, 61, 66, 68, and 82, numbered by capital letters "A" to "F," 
respectively) used in the present experiment, each colony receiving 
from two to four combs. The colonies from which the inserted frames 
were obtained had been dead for from seven weeks to five months 
before they were given to the colonies. None of the 6 colonies were 
strong, the bees being easily accommodated on from four to six 
brood frames, a strength representing about an average for the apiary. 

Out of 110 bees examined from the 6 colonies of the experiment 
prior to the insertion of the combs 10 (9 per cent) were found to be 
infected; and out of 170 bees examined after they were inserted 26 
(15 per cent) were found to be infected. This increase in Nosema- 
infected bees can not be attributed to the introduction of the combs, 
since a similar increase is noted in the other colonies of the apiary 
serving as checks. 

All of the colonies of the experiment lived through the winter and 
spring except one (No. 61). This colony was dead when examined 
in May, 1914. Dead bees taken from the bottom board of the hive 
showed a high percentage of Nosema-infected bees. The 5 colonies 
that survived gained in strength, behaved as healthy colonies, and 
contained a percentage of Nosema-infected bees approximating that 
113789°— 19— Bull. 780 4 



26 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 

of the other colonies of the apiary (Table IV, colonies numbered 36, 
50, 66, 68, and 82). 

The results obtained indicate, therefore, that by inserting combs 
from Nosema-infected colonies, as was done in experiment No. 2, the 
infection is not transmitted appreciably. An explanation for this is 
easily seen from results recorded throughout the present paper. 
Further experiments on the point are summarized in Table XXVI. 

EXPERIMENT NO. 3. 

In this experiment 7 colonies free from Nosema infection were 
inoculated by feeding them sirup to which Nosema apis had been 
added. The bees from which the parasites were obtained for this 
experiment were from various sources (p. 12). They had been dead 
and drying in the laboratory at room temperature for at least three 
months. All of the 7 colonies received the first inoculation feeding 
on October 8. On each succeeding day for four days the feeding 
was repeated. Each of the inoculated colonies of the experiment 
was examined from time to time, but no Nosema-infected bees were 
found. The final examination in connection with this experiment 
was made on October 28. Out of 70 bees examined from the 7 
colonies only one Nosema-infected bee was found. The infection in 
this instance probably did not result from the inoculations. 

In this experiment it is shown that Nosema apis drying in the 
abdomen of bees at room temperature for three months does not 
produce infection when fed to healthy bees. This result suggested 
the interesting fact that the parasite of the bee resisted drying for a 
comparatively short time only (see other experiments, p. 40). 

EXPERIMENT NO. 4. 

In experiment No. 4, four of the colonies used in experiment No. 3 
were inoculated on October 29, 1912, with Nosema apis taken from 
infected bees recently killed. Nine days after the inoculation sam- 
ples of bees were examined from each of the four colonies inoculated. 
Nosema infection was found in nearly all of the bees examined. 
Two weeks after inoculation 50 bees were examined from each of 
the 4 colonies. All of the 200 bees were found to be infected. At 
the end of three weeks a similar condition prevailed. On Decem- 
ber 16, 48 days after inoculation, all of the 4 inoculated colonies 
were alive. A large number of bees were now found on the bottom 
board of the hive. By this time the colonies had become very much 
weakened. The bees were uneasy, the cluster being easily disturbed. 
During the following week 1 of the colonies died out completely. 
The remaining 3 were chloroformed. Another colony inoculated in 
November gave like results, and died in January, 1913. 

Each of the 5 colonies of the experiment were four-framed nuclei. 
As the inoculations were made late in the autumn there were no 



NOSEMA-DISEASE. 27 

young bees emerging. All of the bees of the colonies were exposed, 
therefore, to infection by the inoculation. 

It is seen from this experiment that during the autumn workers 
infected with Nosema apis live, as a rule, for more than one month, 
but that most of them die during the second month after infection. 
These results led to the conclusion that heavy infection in a colony 
when no brood is being reared will destroy the colony, but that it 
may live for two or three months following the infection. 

Although 100 per cent of the workers in each of the 5 colonies 
were infected, the queens from 3 of them were free from infection at 
the death of their respective colonies. The other 2 were found to 
be infected. 

NOSEMA INFECTION WEAKENS THE COLONY. 

There is good evidence at hand indicating that Nosema infection 
weakens the colony. The fact that the epithelial layer of the stomach 
is filled with parasites (fig. 3; Pis. I and II) at once suggests that the 
functions of the organ, digestion and absorption at least, would be 
decidedly impaired thereby. Likewise, when the Malpighian tubules 
are invaded (Pis. II and III), it is to be expected that the bee suffers 
impaired functions. The abnormal condition argues strongly that 
such a bee is less efficient as a member of a colony than an uninfected 
one. Further evidence that infection weakens a colony is seen in 
the fact that in nature the heaviest infection is encountered in the 
weaker colonies. Still further evidence is seen in the results obtained 
in experiments Nos. 1, 3, and 4, just recited, and from inoculations 
made in 1913, 1914, 1915, and 1916, now to be referred to. 

On June 4, 1913, a colony was inoculated by feeding it Nosema 
apis in a sirup suspension. On the 13th it was found to be heavily 
infected. At this time the inoculation was repeated. When exam- 
ined on July 12 the colony had not increased in strength as the unin- 
oculated ones had done. On this date it was reinoculated. By the 
middle of August it had not gained in strength. No reason could 
be assigned for the failure of the colony to become strong other than 
the presence in it of Nosema infection resulting from the inoculation. 

On June 9, 1914, a colony was inoculated with Nosema apis. On 
the 22d it was found to be heavily infected. On July 8 it was 
reinoculated, at which time it was weaker than the check colonies. 
On August 6 the colony was still relatively weak and was reinocu- 
lated. On the 17th it was still weak. The failure on the part of 
the colony to become stronger is attributed to the Nosema infection. 

On August 6, 1914, a colony was inoculated with Nosema apis. 
It became heavily infected and on September 9 it seemed to be 
weakened as a result of the infection. It was reinoculated on this 
date. On December 1 it was found to be heavily infected and on 
January 15, 1915, it was dead. 



/ 



28 BULLETIN 780, U. S. DEPAKTMENT OF AGRICULTUKE. 

On March 30, 1915, a colony was inoculated, resulting in heavy 
infection with Nosema apis. On June 17 the inoculation was repeated. 
Later a swarm was cast. Inoculations were repeated on July 3, 9, 
17, 24, 31, and August 13. The colony became much weakened and 
later in the autumn died. 

Beginning on March 22, 1916, a colony was inoculated at irregular 
intervals thereafter until September. Much brood was being reared 
in it throughout the season, but its strength in September was about 
equal to its strength in March. 

The evidence obtained, it will be observed, is sufficient to justify 
the conclusion that the Nosema infection in a colony tends to weaken 
it. The weakness resulting does not occur immediately following the 
infection, however. During the active brood-rearing season the 
you!ng bees reared may exceed the loss from disease and the colony 
will then actually gain in strength. On comparison of colonies that 
are infected with those that are not, however, it will be seen that the 
infected ones are the weaker. An experimental colony receiving 
repeated inoculations increases in strength, as a rule, during the first 
two weeks following the initial feeding through the emergence of 
young bees, but comparatively little, if any, after the first month. 

The question arises as to whether the weakness is the result of 
infection in workers, drones, or the queen, or in a combination of 
these different members of the colony. Brood apparently does not 
become affected with Nosema apis (p. 10). The weakness in a colony 
can not be attributed, therefore, to infection of the brood. Infection 
among drones is rare (p. 11). Loss in strength, therefore, could not 
be expected to result from infection in the drones. The queen in an 
infected colony is more often free from the infection than not (p. 11). 
Weakness from Nosema infection can result, therefore, when the queen 
is free from infection. By elimination in this way the conclusion is 
reached that the weakness produced by Nosema infection in a colony 
is due primarily to infection among the adult workers. 

Other observations made point to the same conclusion. Workers 
taken from colonies in which Nosema infection had reached a rather 
advanced stage were confined in the Mclndoo wire-screen cages ' and 
kept at room temperature. Healthy ones were similarly caged and 
kept under observation. The relative length of time that the infected 
and uninfected bees lived under these conditions was noted. 

On December 8, 1914, in each of four cages were placed from 15 
to 30 bees taken from colonies heavily infected with Nosema apis. 
By the end of one week, out of 79 bees confined 62 (78 per cent) had 
died. On the same date bees from another infected colony were simi- 
larly confined. At the end of a week out of 119 bees confined 108 
(91 per cent) had died. On December 15, 1914, the experiments were 

i Small triangular cages devised by Mclndoo (1917, p. 4) in his studies on the honeybee. 



NOSEMA-DISEASE. 29 

repeated. Out of 138 bees in one set of four cages 125 (91 per cent) 
were dead at the end of one week. In the other set of four cages out 
of 136 bees confined 98 (72 per cent) were dead at the end of a week. 

On December 8 a check experiment was begun. In each of two 
cages bees taken from healthy colonies were confined and kept at 
room temperature. At the end of one week out of 59 bees confined 
5 (8 per cent) had died. 

Out of a total of 472 diseased bees confined 393 (83 per cent) were 
dead at the end of one week, while out of a total of 59 healthy bees 
kept under similar conditions only 5 (8 per cent) were dead at the 
end of a week. Although such experiments are subject to great 
variation and should be repeated many times for definite results, yet 
the difference between 83 per cent of loss in the case of infected bees 
and 8 per cent of loss in the case of healthy ones is sufficiently great 
to justify the conclusion that the heavily infected bees under the con- 
ditions of the experiment possessed less endurance than the healthy 
ones. These results indicate that weakness in a colony may result 
directly from infection among the workers. 

Throughout the investigations which have been made on the dis- 
ease, therefore, evidence has been obtained indicating that weakness 
results not from the infection of the queen, drones, or brood, but of 
the workers. 

RESISTANCE OF NOSEMA APIS TO HEATING. 

NOSEMA APIS SUSPENDED IN WATER. 

Preliminary results indicating the minimum amount of heating 
that is necessary to destroy Nosema apis were given in an earlier 
paper (White, 1914). Other experiments have been performed. In 
conducting the experiments a suspension was made in water of the 
crushed stomachs and intestines of Nosema-infected bees. This 
suspension was distributed in test tubes in such a dilution that the 
amount in each tube contained the infective material of from 5 to 10 
bees. The tubes were stoppered and heated at different degrees of 
temperature by immersing them in water. Colonies free from infec- 
tion were inoculated with the heated material and the results noted. 

Table VI summarizes some of the experiments made with the 
results obtained. 



30 



BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 



Table VI. — Experiments to determine the heat required to destroy Nosema apis suspended 

in water. 1 



Date of inoculation. 



Temperature 


Period of 


employed. 


heating. 


°C. 


°F. 


Minutes. 


50 


122 


20 


55 


131 


10 


56 


133 


10 


56 


133 


10 


58 


136 


10 


57 


135 


10 


58 


136 


10 


58 


136 


10 


59 


138 


10 


60 


140 


10 


60 


140 


10 


60 


140 


10 


61 


142 


10 


65 


149 


10 


65 


149 


10 


80 


176 


20 


100 


212 


5 



Results of inoculations. 



Jan. 31, 1913.. 
Jan. 8, 1913.. 
Oct. 4, 1913.. 
Oct. 15, 1913. 
May 21, 1915. 
Oct. 15, 1913. 
Feb. 8, 1913.. 
Oct. 4, 1913.. 
Aug. 28, 1915 
Nov. 11, 1912 
Nov. 20, 1912 
May 21, 1915. 
Aug. 28, 1915 
Nov. 12, 1913 
Jan. 8, 1913.. 
Oct. 29, 1912. 
Nov. 12, 1912 



Nosema infection produced. 

Do. 

Do. ■ 

Do. 

Do. 
No infection produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 



1 In omitting fractions of degrees the nearest whole number is given. 



From Table VI it will be observed that Nosema apis in a water sus- 
pension was destroyed in 10 minutes at a temperature somewhere 
between 135° F. (57° C.) and 138° F. (59° C). 



NOSEMA APIS SUSPENDED IN HONEY. 



From preliminary experiments it was learned that the amount of 
heating that is required to destroy Nosema apis suspended in glycerin 
is approximately equal to that required to destroy it when suspended 
in water. It was anticipated, therefore, that the minimum amount 
of heating that would destroy the germ suspended in honey would 
approximate that required to destroy it when suspended in water. 

Experiments were made to determine the approximate thermal 
death point of Nosema apis when it is suspended in honey. In 
making the experiments the technique used was similar in the main 
to that of the preceding group of experiments wherein suspensions in 
water were heated. In Table VII are summarized the experiments 
performed, together with the results obtained. 



Table VII. 



-Experiments to determine the heat required to destroy Nosema apis suspended 
in honey. 



Date of inoculation. 



Aug. 28, 1915 
Aug. 27, 1915 
June 9, 1915. 
May 21, 1915. 
June 8, 1915. 
Aug. 28, 1915 
June 9, 1915 . 
June 8, 1915. 
May 5, 1915.. 
May 21, 1915. 
May 21, 1915. 



Temperature 


Period of 


employed. 


heating. 


°C. 


°F. 


Minutes. 


58 


136 


10 


59 


138 


10 


59 


138 


10 


60 


140 


10 


61 


142 


10 


61 


142 


10 


62 


144 


10 


63 


145 


10 


65 


149 


10 


70 


158 


10 


80 


176 


10 



Results of inoculations. 



Nosema infection produced. 

Do. 
No infection produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 



NOSEMA-DISEASE. 



31 



Table VII shows that Nosema apis in a honey suspension was 
destroyed by heating for 10 minutes at a temperature between 136° F. 
(58° C.) and 140° F. (60° C), the death point being about 138° F. 
(59° C). 

RESISTANCE OF NOSEMA APIS TO DRYING. 

In experiments relative to the effect of drying on Nosema apis, 
stomachs from Nosema-infected bees were crushed, and the crushed 
tissues were smeared on slides to the extent of a thin layer. The 
slides were placed in incubator, room, outdoor, and refrigerator tem- 
peratures, respectively. At different intervals after the preparation 
of the smears an aqueous suspension was made, germs from two slides 
representing the material from 5 to 20 bees being used. This was 
added to sirup and fed to a healthy colony. Whether or not the 
parasite had been destroyed was determined by the presence or 
absence of Nosema-infection in the colony following the inoculation 
with the sirup. 

NOSEMA APIS DRYING AT INCUBATOR TEMPERATURE. 

In Table VIII are summarized the experiments, together with the 
results obtained, in which the Nosema material was allowed to dry at 
incubator temperature. 

Table VIII. — Resistance of Nosema apis to drying at incubator temperature. 



Date of inoculation. 



July 30, 1915. 
July 14, 1916. 
Oct. 5,1914.. 
July 21, 1915. 
July 29, 1916. 
Sept. 11, 1914 
Nov. 2, 1914.. 
Sept. 29, 1914 
Oct. 16, 1914. 
July 9, 1915.. 
May 24, 1915 . 
July 9, 1915.. 



Period of drying. 



Months. 









2 
7 
9 



Days. 



Results of inoculation. 



Nosema infection produced. 

Do. 

Do. 

Do. 
No infection produced. 

Do. 

Do. r 

Do. 

Do. 

Do. 

Do. 

Do. 



From Table VIII it will be seen that Nosema apis drying at incu- 
bator temperature was destroyed in from 15 to 21 days, that is, 
during the third week. 

NOSEMA APIS DRYING AT ROOM TEMPERATURE. 

In Table IX are summarized experiments in which the drying of 
Nosema apis took place at room temperature. 



32 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 

Table IX. — Resistance of Nosema apis to drying at room temperature. 



Date of inoculation. 




Results of inoculation. 



July 26, 1916. 
Sept. 11, 1914 
Aug. 11, 1916 
Oct. 2, 1914.. 
Sept. 1,1915. 
Aug. 26, 1916 
Oct. 16,1914. 
May 24, 1916. 
Sept. 1,1915.. 
June 27, 1916. 



Nosema infection produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 
No infection produced. 

Do. 

Do. 



From results recorded in Table IX it will be observed that Nosema 
apis drying at room temperature remained virulent for from 56 to 
60 days, that is, about 2 months. 

NOSEMA APIS DRYING AT OUTDOOR TEMPERATURE. 

Table X summarizes experiments in which Nosema apis was allowed 
to dry at outdoor temperature. 

Table X. — Resistance of Nosema apis to drying at outdoor temperature. 



Date of inoculation. 



Period of drying. 


Months. 


Days. 





21 





42 





46 





56 





60 





60 





75 





80 





85 





100 


9 





10 


10 


11 






Results of inoculation. 



Sept. 11, 1914 
Oct. 2, 1914.. 
Aug. 17, 1914. 
Oct. 16, 1914. 
June 7, 1916.. 
Sept. 27, 1913 
July 9, 1915.. 
June 27, 1916. 
Aug. 17, 1915. 
July 17, 1916. 
May 25, 1915. 
July 17, 1914. 
July 1,1914.. 



Nosema infection produced. 

Do. 

Do. 

Do. 

Do. 
No infection produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 



The results recorded in Table X show that Nosema apis ceased to 
be virulent after 2 months of drying at outdoor temperature. 

NOSEMA APIS DRYING AT REFRIGERATOR TEMPERATURE. 

Table XI summarizes experiments in which Nosema apis was 
allowed to remain dry in the refrigerator. 

Table XI. — Resistance of Nosema apis to drying at refrigerator temperature. 1 



Date of inoculation. 



Period of 
drying. 




Results of inoculation. 



Dec. 2, 1915. 
Jan. 3, 1916 . 
Mar. 3, 1916. 
Apr. 2, 1916. 
Apr. 22, 1916 
May 3, 1916. 
July 3, 1916. 



Nosema infection produced. 

Do. 

Do. 

Do. 
No infection produced. 

Do. 

Do. 



1 A few times during the experiments in which the refrigerator temperature was used, the ice became 
exhausted, allowing the temperature to approach and possibly to reach that of the room. This higher 
temperature, when present, however, at no time prevailed for more than a day. 



NOSEMA-DISEASE. 



33 



It is learned from the results recorded in Table XI that Nosema 
apis drying at refrigerator temperature remained virulent for seven 
months but that no disease was produced following inoculation with 
the material after seven and one-half months drying. 

From the results obtained in the experiments relative to the resist- 
ance of Nosema apis to drying, given in Tables VIII-XI, it will be 
observed that the period the parasite remained alive, or at least 
virulent, varied, depending upon the environment of the germ. The 
shortest period for the destruction of spores was obtained under 
incubator conditions, while the longest period occurred under refrig- 
erator conditions. The death probably was not due to the drying 
alone but to a combination of factors of which drying was an impor- 
tant one. 

RESISTANCE OF NOSEMA APIS TO FERMENTATION. 

Experiments have been made to obtain data relative to the resist- 
ance of Nosema apis to fermentative processes. In conducting the 
experiments suspensions of the crushed stomachs from Nosema- 
infected bees were made in a 10 per cent sugar (saccharose) solution 
and in a 20 per cent honey solution. These solutions were distributed 
in test tubes. Each tube contained infectious material equal to that 
present in the stomachs of from 5 to 10 infected bees. To each sus- 
pension was added a bit of soil to inoculate it further. Suspensions 
were allowed to ferment at incubator, room, outdoor, and refrigerator 
temperatures, respectively. At intervals reckoned in days the fer- 
menting suspension from a single tube was transferred to about one- 
half pint of sugar sirup and fed to a colony free from the infection. 
The results were then noted. 

FERMENTATION AT INCUBATOR TEMPERATURE. 

In Table XII are summarized some of the results that were obtained 
when a suspension of Nosema apis in a 20 per cent aqueous solution 
of honey was allowed to ferment at incubator temperature. 

Table XII. — Resistance of Nosema apis to fermentation in a honey solution. 



Date of inoculation. 



Period of 
fermen- 
tation. 



Results of inoculation. 



July 25, 1916 
July 26, 1916 
July 27, 1916 
July 28, 1915 
July 12, 1915 
July 15, 1916 
July 17, 1916 



Days. 

2 
3 
4 
5 
8 
10 



Nosema infection produced. 

Do. 
No infection produced. 

Do. 

Do. 

Do. 

Do. 



34 



BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 



From experiments recorded in Table XII it was shown that Nosema 
apis was destroyed by fermentation in 20 per cent honey solution at 
incubator temperature in three days. 



FERMENTATION AT ROOM TEMPERATURE. 

In Table XIII are summarized experiments in which colonies were 
inoculated with a suspension of Nosema apis in a 10 per cent sugar 
(saccharose) solution, which had been allowed to ferment, at room 
temperature. 

Table XIII. — Resistance of Nosema apis to fermentation in sugar solution at room 

temperature. 



Date of inoculation. 



Time of fermenta- 
tion. 



Results of inoculation. 



Sept. 8, 1915. 
Sept. 9, 1915. 
June 4, 1915.. 
Sept. 10, 1915 
July 27, 1915. 
July 29. 1915. 
Sept. 13, 1915 
Sept. 15, 1915 
Sept. 16, 1915 
Sept. 1,1915. 
Jan. 9, 1915.. 
Sept. 15, 1914 
Sept. 29, 1914 
June 9, 1914.. 
June 10, 1914. 
May 13, 1915. 



Months. 






, 






7 
10 
18 



Bays. 



Nosema infection produced. 

Do. 
No infection produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 



From Table XIII it will be seen that the parasite was destroyed 
by fermentation in a 10 per cent sugar solution at room temperature 
in from 7 to 1 1 days. The range of variation shown may be attributed 
largely to variation in the temperature. 

FERMENTATION AT OUTDOOR TEMPERATURE. 

In Table XIV are summarized experiments made for the purpose 
of obtaining approximate data relative to the resistance of Nosema 
apis in a 20 per cent honey solution at outdoor temperature. 

Table XIV. — Resistance of Nosema apis to fermentation in a honey solution at outdoor 

temperature. 



Date of inoculation. 



Period of 
fermen- 
tation. 




Results of inoculation. 



July 26, 1916. 
July 27, 1916. 
July 28, 1916. 
Sept. 8, 1915. 
July 29. 1916. 
Aug. 30, 1916 
July 29, 1916. 
Aug. 31, 1916 
Sept. 2, 1916. 
Sept. 6, 1916. 



Nosema infection produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 
No infection produced. 

Do. 



NOSEMA-DISEASE. 



35 



From Table XIV it will be observed that the parasite was destroyed 
in 9 days in the presence of fermentation processes taking place in 
a 20 per cent honey solution at outdoor temperature. 

At refrigerator temperature it was found that Nosema apis resisted 
fermentative processes for more than seven and less than nine days. 

It will be observed from the results obtained that Nosema apis 
in the presence of fermentative processes is destroyed in a compara- 
tively short time. The period, it will be seen, varies somewhat with 
the temperature of the fermenting suspension. The experiments 
tend to indicate, furthermore, that the time element depends slightly 
upon the nature of the fermenting medium, the germ being destroyed 
sooner in a honey solution than in a saccharose one. The time element 
is dependent also upon the strength of the solutions employed. 

RESISTANCE OF NOSEMA APIS TO PUTREFACTION. 

Experiments have been made for the purpose of obtaining results 
relative to the resistance possessed by Nosema apis to putrefactive 
processes. The nature of the experiments was similar to those rela- 
tive to fermentation but instead of sugar solutions used for the 
suspensions a 1 per cent peptone solution in water was employed. 
In the experiments, suspensions, after undergoing putrefactive changes 
at incubator, room, outdoor, and refrigerator temperatures, respec- 
tively, were used in the inoculation of colonies. 

PUTREFACTION AT INCUBATOR TEMPERATURE. 

The experiments summarized in Table XV indicate the resistance 
of Nosema apis to putrefaction at incubator temperature. 

Table XV. — Resistance of Nosema apis to putrefaction at incubator temperature. 



Date of inoculation. 



Period of 
putrefac- 
tion. 



Results of inoculations. 



July 25, 1916. 
July 26, 1916. 
July 27, 1916. 
July 28, 1916. 
July 12, 1916. 
Sept. 10, 1915 
July 15, 1916. 
July 17, 1916. 



Days. 



Nosema infection produced. 

Do. 

Do. 

Do. 
No infection produced. 

Do. 

Do. 

Do. 



By the results recorded in Table XV it is shown that Nosema 
apis was destroyed by putrefaction at incubator temperature in 
five days. 



36 



BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 



PUTREFACTION AT ROOM TEMPERATURE. 



In Table XVI are summarized experiments in which the putrefac- 
tive processes took place at room temperature. 

Table XVI, — Resistance of Nosema apis to putrefaction at room temperature. 



Date of inoculation. 



Period of 
putrefac- 
tion. 



Results of inoculation. 



July 28, 1915. 
July 21, 1915. 
July 28, 1915. 
Sept. 29, 1914 
July 1,1915.. 
Aug. 20, 1914 



Days. 



Nosema infection produced. 
No infection produced. 

Do. 

Do. 

Do. 

Do. 



From Table XVI it is seen that Nosema apis at room temperature 
resisted the putrefactive processes for about two weeks. As the 
room temperature varies it is to be expected that the time required 
for the destruction of the parasite will vary also. 

PUTREFACTION AT OUTDOOR TEMPERATURE. 

The following table summarizes experiments that indicate the 
period Nosema apis resists putrefaction at outdoor temperature: 

Table XVII. — Resistance of Nosema apis to putrefaction at outdoor temperature. 



Date of inoculation. 



Period of 
putrefac- 
tion. 



Results of inoculation. 



July 26, 1916. 
July 27, 1916. 
July 28, 1916. 
July 29, 1916. 
Aug. 31, 1916 
Sept. 2, 1916. 
Sept. 6, 1916. 
Aug. 26, 1916 
Sept. 2, 1916. 



Bays. 



Nosema infection produced. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 



In the experiments recorded in Table XVII it will be observed that 
Nosema apis was not destroyed in the presence of putrefactive changes 
at outdoor temperature in 22 days. 

At refrigerator temperature the parasite has resisted putrefaction 
for more than three months. 

The foregoing experiments relative to the effect of putrefactive 
processes on Nosema apis show that the parasite may be destroyed as 
a result of putrefaction. They show also that the temperature of the 
suspension is a factor in determining the period of resistance. Further- 
more, it is seen that the germ resists the destructive processes accom- 
panying putrefaction longer than those accompanying fermentation. 



XOSEMA-DISEASE. ' 37 

RESISTANCE OF NOSEMA APIS TO DIRECT SUNLIGHT. 

RESISTANCE WHEN DRY. 

Petri dishes (fig. 6) which were smeared with the crushed stomachs 
of Xosema-infected bees were exposed to the direct rays of the sun. 




Fig. 6. — Open Petri dish. One-half of the dish, either top 
or bottom. 

After intervals reckoned in hours healthy colonies were inoculated 
with a suspension made from the dishes which had been exposed. 
Table XVIII summarizes the experiments and the results obtained. 

Table XVIII. — Resistance of Xosema apis when dry to direct sunlight. 



Date of inoculation. 



Period of 
exposure 
to sun. 



Results of inoculation. 



Aug. 2, 1915 

Aug. 21, 1914 

Aug. 2, 1915 

July 28, 1915 

Aug. 27, 1914 

Aug. 23, 1915 

Aug. 20, 1915 • 

Sept. 2, 1914 

Aug. 25, 1915 

Sept. 3, 1914 

Sept. 14, 1915 ' 

Aug. 17,1915 

Sept. 10, 1915 

Aug. 19, 1915 

Sept. 11,1915 

Aug. 24, 1914 

Sept. 13, 1915 

Aug. 4. 1914 

Sept. 16, 1915 



Hours 



2 Xosema infection produced. 

5 Do. 

5 Do. 

8 Do. 

10 Do. 

13 Do. 

15 Do. 

15 Do. 

17 Do. 

20 Do. 

29 Do. 

15 No infection produced. 



i; 

is 

21 
22 
32 
34 
35 



Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 



The results hi Table XVIII show that Nosema apis was destroyed 
in the experiments recorded in from 15 to 32 hours' exposure to direct 
sunlight. 

It will be readily appreciated that the time that Nose ma apis will 
resist the destructive effects of the sun's rays will vary largely accord- 
ing to the intensity of the rays, the heat present, and the thickness of 
the layer of infective material exposed. 



DESTRUCTION IN WATER. 



In experiments made for the purpose of determining the time re- 
quired to destroy Nosema apis suspended in water, an aqueous 
suspension of the crushed stomachs of about 10 bees was poured into 
each of a number of Petri dishes (fig. 6) and exposed to the direct 



38 



BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 



rays of the sun. The top of the dish was not on during the exposure. 
After intervals reckoned in hours inoculations were made of healthy 
colonies, the germs contained in one dish being used. 

Table XIX gives a summary of a set of experiments of this kind. 

Table XIX. — Resistance of Nosema apis suspended in water to the direct rays of the sun. 



Date of inoculation. 



Period of 
exposure. 



Results of inoculation. 



Aug. 2, 1915.. 
July 27, 1915. 
Aug. 20, 1915. 

Do 

Aug. 26, 1915. 
Sept. 10, 1915, 
Aug. 27, 1915. 
Sept. 11,1914, 
Sept. 13, 1915, 

Do 

Sept. 14, 1915 
Sept. 16, 1915. 

Do 

Sept. 17, 1915. 



Hours. 
2 

10 
12 
18 
20 
20 
27 
27 
44 
37 
51 
58 
65 
72 



Nosema infection produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 
No infection produced. 

Do. 

Do. 

Do. 

Do. 



The results in the foregoing table show that Nosema apis was de- 
stroyed by the direct rays of the sun in from 37 to 51 hours. It is 
seen, therefore, that Nosema apis when suspended in water shows a 
considerable amount of resistance. In the question of the transmis- 
sion of the disease this resistance may be of considerable importance. 

At the time these experiments were made the intensity of the rays 
was, as a rule, quite marked and, therefore, favorable for the destruc-^ 
tion of germs. The temperature of the aqueous suspension, however, 
did not reach 136° F. (58° C.) and, therefore, was not sufficient to 
destroy the virus through heating. Some of the suspensions stood for 
more than a week in the Petri dishes, thereby introducing the factors 
of fermentation and putrefaction. The effect of these factors on the 
results is not known. 

DESTRUCTION IN HONEY. 

In performing the experiments crushed stomachs from about 10 
Nosema-infected bees were suspended in about 3 ounces of honey in 
Petri dishes (fig. 7). To prevent robbing by bees the dish was used 
with the top on. The suspension was exposed to the direct rays of the 
sun with the dishes resting on a wooden support. After different 
intervals healthy colonies were inoculated with germs which had 
been exposed to the sun. 

Even when resting on a wooden support it is not unusual during the 
summer for the honey of the suspension exposed to the sun to reach a 
temperature beyond the thermal death point of the parasite. To 
determine facts in regard to the effect of the sun's rays on Nosema 
apis, therefore, this point in regard to heat must be met by the 
technique employed. This could have been done quite easily but 
for the lack of time. 



NOSEMA-DISEASE. 



39 



In the experiments it was found that Nosema apis was destroyed 
in all instances in which the temperature of the honey reached or 
exceeded 140° F. (60° C), a temperature at which the germ is killed 
by heat (p. 30). Sufficient data, therefore, have not been obtained 
to warrant a definite conclusion regarding the time required for the 




F!G. 7.— Petri dish. The top half is slightly raised. Those 
used here are 4 inches in diameter. 

direct sunlight to destroy Nosema apis suspended in honey. The 
results obtained from the experiments made in which aqueous sus- 
pensions were exposed to the sun give some idea as to the probable 
approximate time which would be required. 

PERIOD NOSEMA APIS REMAINS VIRULENT. 

PERIOD IN HONEY. 

In experiments made to determine the length of time Nosema apis 
remains virulent in honey a suspension of the parasite in honey was 
distributed in flasks, about one-half pint being poured into each flask. 
These were placed at room temperature and shielded from the light. 
After different intervals colonies were inoculated, the suspension 
from a single flask being used. The results obtained are included 
in Table XX. 

Table XX. — Period Nosema apis remains virulent in honey. 



Date of inoculation. 



Period in honey. 



Results of inoculation. 



Oct. 20, 1914. 
Feb. 4, 1915.. 
Feb. 24, 1915. 
Feb. 4, 1915.. 
Jan. 16, 1915. 
July 14, 1915. 
July 23, 1915. 
Oct. 21, 1915. 
June 11, 1915 
Sept. 3, 1915. 
June 24, 1915. 
Oct. 21, 1915. 
July 24, 1916. 
Aug. 14, 1913 
May 1, 1915. . 
June 9, 1914. 
Apr. 27, 1915. 
May 5, 1914.. 
July 26, 1916. 



Months. 
1 
1 
2 
3 
3 
2 
2 
2 
3 
3 
3 
3 
4 
5 
7 
7 
9 
9 
12 



Days. 


18 


10 
27 

6 
15 
25 

5 
17 
20 
21 

4 


17 
19 

7 
19 





Nosema infection produced. 

Do. 

Do. 

Do. 

Do. 
No infection produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 



40 



BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 



The experiments summarized in Table XX show from the results 
recorded that Nosema apis, when suspended in honey and kept at 
room temperature, shielded from the light, remained virulent for 
from 66 to 124 days, that is, from 2 to 4 months. The wide varia- 
tion noted here probably is due very largely to the variation in 
temperature of the honey suspension. 

PERIOD IN DEAD BEES. 

Among the factors tending to destroy Nosema apis within the 
remains of dead bees are drying, putrefaction, and probably fermen- 
tation. The temperature also is to be expected to vary the period 
of resistance. In conducting the experiments, therefore, incubator, 
room, outdoor, and refrigerator temperatures were used. Infected 
bees were killed and kept in these different environments. After 
different intervals suspensions were made in sirup, the crushed 
bodies of from 5 to 10 of the infected bees being used. Colonies 
were inoculated with the suspensions. 

AT INCUBATOR TEMPERATURE. 

Table XXI summarizes the results obtained when inoculations 
were made with suspensions of Nosema-infected material from bodies 
of bees kept at incubator temperature. 

Table XXI. — Resistance of Nosema apis within dead bees at incubator temperature 

(37.5° C). 



Date of inoculation. 



Period 

of 
drying. 



Results of inoculation. 



Apr. 9, 1916. . 
Apr. 12, 1916. 
Apr. 14, 1916. 
June 27, 1916 
July 1,1916.. 
May 17, 1916. 
Aug. 4, 1915. 
Oct. 8, 1914.. 
Aug. 8, 1915. 
Aug. 17, 1915 
Oct. 19, 1915. 
Aug. 23, 1915 
Aug. 6, 1914. 
Nov. 2, 1914. 



Days. 



Nosema infection produced. 

Do. 

Do. 
No infection produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 



By the results recorded in the experiments summarized in Table 
XXI, it was shown that Nosema apis in the bodies of dead bees 
kept at incubator temperature ceased to be virulent in less than one 
week. 



AT ROOM TEMPERATURE. 



In Table XXII are summarized the experiments in which dead 
kept at room temperature, furnished the Nosema-infected 
material for the suspensions used in the inoculations. 



NOSEMA-DISEASE. 41 

Table XXII. — Resistance of Nosema apis in dead bees kept at room temperature. 



Date of inoculation. 



Aug. 4, 1915 . 
Aug. 10, 1915 
July 17, 1916. 
Aug. 26,1916 
Aug. 17, 1916 
June 3, 1916. 
Aug. 23, 1915 
July 26, 1916 
Aug. 1,1916. 
June 27, 1916 
Aug. 20, 1914 
Aug. 30, 1914 




Results of inoculation. 



Nosema infection produced. 

Do. 

Do. 

Do. 
No infection produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 



From Table XXII it is learned that when dead infected bees were 
kept at room temperature the parasite remained virulent for three 
or four weeks, but did not produce the disease after one month. 
Since the temperature of the room was not constant, variations in 
results obtained at this temperature are to be expected. 

AT OUTDOOR TEMPERATURE. 

Dead Nosema-inf ected bees were placed in a hive body standing in the 
experimental apiary. At different intervals suspensions were made 
and colonies were inoculated. In Table XXIII are summarized a 
few experiments indicating by the results obtained the approxi- 
mate period Nosema apis remains virulent in the body of dead bees 
at outdoor temperature. 

Table XXIII. — Resistance of Nosema apis in dead bees drying at outdoor temperature. 



Date of inoculation. 




Results of inoculation. 



Oct. 19, 1914. 
Aug. 23, 1915 
Nov. 2, 1914. 
June 7, 1916. 
June 27, 1916 
July 17,1916. 



Nosema infection produced. 

Do. 

Do. 
No infection produced. 

Do. 

Do. 



From Table XXIII it is seen that Nosema apis in the bodies of 
dead infected bees kept dry at outdoor temperature remained viru- 
lent for from five to six weeks. These experiments extended over a 
period from June to November, as shown by the dates. It is to be 
expected that if they had been conducted throughout the year the 
results obtained would have shown a much wider range of variation. 



42 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 



AT REFRIGERATOR TEMPERATURE. 



In Table XXIV are summarized experiments the results of which 
indicate the approximate period during which Nosema apis remains 
virulent in the bodies of infected bees kept at refrigerator temper- 
ature. 

Table XXIV. — Resistance of Nosema apis in dead bees drying at refrigerator temperature. 



Date of inoculation. 


Period in 
refrig- 
erator. 


Results of inoculation. 


Mar 4, 1916. \ j 


Months. 
2 
2 
2i 
3 
3§ 
3 
4 
4 
4 
5 
6 
8 
10 


Nosema infection produced. 
Do. 


Mar. 20, 1916 


Mar. 20, 1916 


Do. 


Apr. 22,1916 


Do. 


May 6, 1916 


Do. 


Dec. 7, 1915 


No infection produced. 
Do. 


Jan. 7, 1916 


May 24, 1916 , 


Do. 


June 3, 1916 


Do. 


Feb. 10, 1916 


Do. 


Apr. 7, 1916 


Do. 


May 6, 1916 


Do. 


July 3, 1916 


Do. 







The results recorded in Table XXIV show that Nosema apis in 
the bodies of dead infected bees remained virulent at refrigerator 
temperature from two and a half to four months. 

ON THE SOIL. 

Dead Nosema-infected bees were placed on the soil in the open, 
but in a somewhat shaded spot. After different intervals of time 
colonies were inoculated, these dead bees being used as the source 
for the infective material. Table XXV summarizes the experi- 
ments performed, the results of which indicate the approximate 
period during which Nosema apis remains virulent in the bodies of 
dead bees lying on the soil. 

Table XXV. — Resistance of Nosema apis in dead bees lying on the soil. 



Date of inoculation. 



Period 
on soil. 



Results of inoculation. 



July 16, 1915. 
Aug. 1,1916. 
Aug. 28, 1915 
Aug. 12, 1916 
Aug. 26, 1916 
Aug. 28, 1915 
May 14, 1916. 
Oct. 4, 1915.. 
Oct. 21, 1915. 
Oct. 21, 1915. 
Oct. 4, 1915.. 
Nov. 9, 1915. 



Days. 
13 
18 
25 
29 
43 
44 
71 
77 
85 
94 
104 
104 



Nosema infection produced. 

Do. 

Do. 

Do. 

Do. 

Do. 
No infection produced. 

Do. 

Do. 

Do. 

Do. 

Do. 



NOSEMA-DISEASE. 



43 



From the results recorded in Table XXV it is seen that when the 
dead Nosema-inf ected bees were allowed to remain on the soil exposed 
to outdoor conditions Nosema apis was virulent at the end of 44 days, 
but the germ had lost its virulence before 71 days. Results of 
experiments having the nature of those referred to in this table 
naturally depend largely upon the climatic conditions which prevail. 

It was observed that insects, ants especially, fed upon the dead 
bees lying on the ground. In this way they removed much of the 
material containing the parasites. This fact must be borne in mind 
in a consideration of the length of time that bees dead of Nosema 
disease and lying on the soil might serve as a possible source of 
infection. 

The five foregoing groups of experiments relative to the period 
during which Nosema apis remains virulent in the bodies of dead 
infected bees show that the period varies with the environment under 
which the bees are kept, the temperature being an important factor 
in causing the variation. It is interesting to note that under fairly 
favorable conditions for its preservation Nosema apis remains 
virulent within the bodies of dead infected bees only three months, 
while under less favorable conditions its destruction occurs in a much 
shorter period. 

INFECTIOUSNESS OF BROOD-COMBS FROM NOSEMA-INFECTED 

COLONIES. 

Experiments have been made for the purpose of obtaining data 
relative to the likelihood of the transmission of Nosema disease from 
colony to colony through the medium of brood-combs. Brood- 
combs on which colonies had died of the disease and others taken 
from colonies heavily infected with Nosema apis through experi- 
mental inoculation were inserted into healthy colonies after different 
periods of time had elapsed following their removal. Table XXVI 
gives a summary of experiments made and the results obtained. 

Table XXVI. — Results from insertion of brood-combs from Nosema-inf ected colonies 

into healthy ones. 



Date combs were inserted. 



Period combs 
were stored. 



Number 
of combs 



Results of inoculation. 



Apr. 20, 1915. 



Do 

Apr. 26, 1915. 
July 3, 1915.. 
May 19, 1916. 
Apr. 26, 1915. 

Do 

Apr. 24, 1915. 
June 18, 1914. 
June 29, 1914. 
Apr. 24, 1915. 
May 1,1915.. 
Aug. 22, 1914. 
May 19, 1916. 



Inserted im- 
mediately. 

do 

do 

....do 



\ month.. 
do... 

1 month. . 

2 months. 
....do... 

3 months. 
....do... 

4 months. 
6 months. 



No Nosema infection pro- 
duced. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 



44 



BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 



As will be observed from Table XXVI, infection did not occur in 
any of the experiments in which brood-combs from Nosema-infected 
colonies were given to healthy ones. The practical import of the 
results is that brood-combs from Nosema-infected colonies need not be 
destroyed, but maybe inserted without treatment into hives contain- 
ing healthy bees with practically no fear that losses will result from 
such manipulation. (See also experiment No. 2, p. 25.) 

RESISTANCE OF NOSEMA APIS TO CARBOLIC ACID. 

Stomachs taken from Nosema-infected bees were crushed and 
suspended in aqueous solutions of carbolic acid (commercial) . One, 2, 
and 4 per cent solutions were used. These suspensions, respectively, 
were distributed in test tubes and were allowed to stand at room 
temperature. After different intervals healthy colonies were inocu- 
lated, the suspension from a single tube being used for each. 

A summary of experiments performed with the results obtained is 
given in Table XXVII. 

Table XXVII. — Effect of carbolic acid on Nosemaapis. 



Date of inoculation. 



Per cent 
of car- 
bolic- 
acid 

solution. 



Period in carbolic 
acid. 



Results of inoculation. 



Aug. 18, 1915 
July 16, 1915. 
July 2, 1915.. 
June 9, 1915. 
Aug. 18, 1915 
July 16, 1915. 
July 2, 1915.. 
June 8, 1915. 
July 16, 1915. 
July 2, 1915.. 
June 8, 1915. 



Hours. 



Minutes. 

10 




10 









No infection produced. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 



From the preliminary results given in Table XXVII it will be noted 
that Nosema apis is rapidly destroyed in 1, 2, and 4 per cent aqueous 
solutions, respectively, of carbolic acid, showing that the parasite 
possesses very slight resistance to the disinfectant. 

EFFECT OF DRUGS ON NOSEMA-DISEASE. 



It is natural that beekeepers should have thought of drugs and 
employ them in the treatment of Nosema infection. Preliminary 
experiments have been made to obtain data relative to the effect of 
betanaphthol, salol (phenyl salicylate), carbolic acid (phenol), salicylic 
acid, formic acid, oil of eucalyptus, and quinin (bisulphate of 
quinin) on this infection. It will be recalled that most of these 
drugs have been given a trial from time to time by beekeepers in the 
treatment of one or more of the bee diseases. 



NOSEMA-DISEASE. 



45 



In the experiments honey was diluted with an equal quantity of 
water and medicated. 1 To the medicated solution Nosema apis was 
added. This suspension was fed to a colony, usually within a half 
hour from the time it was made. On each of four or five days imme- 
diately following the inoculation, the colony was fed honey medicated 
with the drug but free from Nosema apis. 

In Table XXVIII are summarized the experiments performed, 
together with the results obtained. 

Table XXVIII. — Effect of drugs on Nosema infection. 



Drug. 


Experiment 1. 


Experiment 2. 


Experiment 3. 


Experiment 4. 


Propor- 
tion. 


Results. 


Propor- 
tion. 


Results. 


Propor- 
tion. 


Results. 


Propor- 
tion. 


Results. 


Betanaphthol 

Salol 


2:1,000 

2:1,000 
2:1,000 
3:1,000 
3:1,000 
5:1,000 
10:1,000 


No infec- 
tion. 

...do 

...do 

...do 

...do 

Infection. 
...do 


1:1,000 

1:1,000 
1:1,000 
2:1,000 
2:1,000 
4:1,000 
4:1,000 


No infec- 
tion. 

...do 

...do 

...do 

...do 

Infection. 
...do 


1:2,000 

1:2,000 
1:2,000 
1:1,000 
1:1,000 
2:1,000 
2:1,000 


No infec- 
tion. 
...do 

Infection. 

...do 

...do 

...do 

...do 


1:5,000 

1:5,000 
1:5,000 


Infection. 
Do. 


Salicylic acid 

Carbolic acid 

Formic acid 

Eucalyptus 

Quinin 


Do. 







The results recorded in Table XXVIII show that the parasite was 
destroyed by some of the drugs used but that it resisted others. 
Their relative efficiency as indicated from these preliminary results 
is shown by the arrangement in the table. Betanaphthol and salol 
seem to be the most effective of those tried, and eucalyptus and 
quinin the least efficient. 

Experiments were performed in which the inoculation with Nosema 
apis was not followed by feedings with medicated sirup. The 
results obtained show that under the conditions of the experiments 
the drugs affected the parasite as seen by the lower percentage of 
Nosema-infected bees in the colonies inoculated. In colonies re- 
ceiving subsequent feedings of medicated sirup a still lower percent- 
age of infected bees was found. 

While it is thus established that Nosema apis is somewhat sus- 
ceptible to the effects of some of the drugs, the experiments are 
altogether too few for definite conclusions as to the extent of their 
action. Statements regarding the effect of the drugs on Nosema- 
disease, therefore, should be accepted cautiously, for the present at 
least, unless they are supported by experimental or other good 
evidence. 



1 In obtaining the desired proportion of the drug, betanaphthol, salol, salicylic acid, and eucalyptus were 
dissolved in alcohol. In the case of carbolic acid, formic acid, and the bisulphate of quinin aqueous 
solutions of the drugs were employed. 



46 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 

MODES OF TRANSMISSION OF NOSEMA-DISEASE. 

No problem in the study of Nosema-disease is more important than 
that of its transmission. The problem is at the same time one of 
the most difficult for complete solution. While further information 
is still much desired, yet it is possible from the studies which have 
been made to arrive at certain conclusions concerning the manner 
in which the disease is spread. The discussions which follow are 
based chiefly upon observations noted in the foregoing pages. 

It is naturally safe to conclude that the transmission of Nosema- 
disease depends directly upon the transmission of the parasite that 
causes it. If the course of Nosema apis in nature were followed 
completely, therefore, the problem relative to the spread of the disease 
would be solved. Such a task is difficult, as the possible sources 
for the parasite and the accompanying conditions are various. 

The fact, determined experimentally, that a suspension of Nosema 
apis in sirup when fed to bees will produce the disease shows quite 
conclusively that infection takes place through the ingestion of the 
parasite. At present there is no evidence that it takes place other- 
wise than by way of the alimentary tract. This leads to the im- 
portant tentative conclusion that the transmission of the disease is 
effected through either the food or the water supply of bees, or both. 

On reaching the stomach by ingestion the parasite begins its growth, 
invades the walls of the organ, multiplies enormously, and forms 
spores which are shed into the lumen and passed out of the alimentary 
tract with the excrement. The chances that any single parasite once 
outside the bee will be ingested and cause infection are very slight. 
The immense number that are produced, however, increases the 
chances very greatly. Again, the chances of infection are very much 
reduced by the many destructive agencies in nature encountered by 
the parasite. Among these are drying (p. 31), heat (p. 29), direct 
sunlight (p. 37), fermentation (p. 33), and putrefaction (p. 35). 

The excrement is voided normally during flight and most often 
soon after the bee leaves the hive. Should the droppings from in- 
fected bees fall into a body of water, such water would become 
thereby contaminated with the Nosema parasite and the use of it 
by bees would expose them to infection. Should the body of water 
be a rapidly flowing one, naturally the chances that other colonies 
of the apiary might become infected from such a source would be 
less than if it were a sluggish one. Should such contaminated water 
be exposed to the sun, the rays of the latter would have a tendency 
to destroy the parasites. The resistance of Nosema apis to the de- 
structive effects of the sun's rays (p. 38) are sufficiently great, 
however, that there would still remain a strong likelihood that infec- 
tion might take place from the water supply. While in the water 



NOSEMA-DISEASE. 47 

the parasites may be subjected to fermentation or putrefaction or 
both. These factors would tend to destroy the germ, although its 
resistance under these conditions is again considerable. 

It has been suggested by some writers that drops of water from 
showers or dew on vegetation about the apiary might become con- 
taminated by excrement present and thus be a source of infection. 
This would seem to be a possibility. The extent, if any, to which 
the disease is thus transmitted is not yet known. 

Should the excrement of infected bees fall on the soil, the chances, 
ordinarily, would be slight that the contained parasite would reach 
a bee and infect it. Should the surface water resulting from rains 
carry the germ into a water supply used by bees, the chances of 
infection from the soil as a source would be considerably increased 
thereby. If the bodies of dead Nosema-infected bees were washed 
into the water supply, contamination of it might follow. 

In estimating the probable danger of infection from the bodies of 
bees dead of Nosema-disease, the possibility of the parasites being 
destroyed after the death of such bees through putrefaction (p. 35), 
drying (p. 31), or other means must be given due consideration. 

The facts which are known concerning Nosema-disease indicate 
that the disease may be transmitted: (1) From the infected bees of 
a colony to healthy bees of the same colony, and (2) from the infected 
bees of a colony to healthy bees of another colony. When the infec- 
tion is transmitted from infected bees to noninfected ones of the 
same colony, the question arises as to whether such infection takes 
place while the bees are within or without the hive. The fact that 
the heaviest infection with Nosema apis occurs in the spring of the 
year, and the further fact that only a comparatively few colonies of 
the apiary are likely to be heavily infected, support the tentative 
conclusion that the transmission of the germ takes place within the 
hive rather than from a source outside of it. 

There are facts concerning the disease, however, which indicate 
that the infection under certain circumstances is not readily trans- 
mitted within the hive. For example, colonies which in the spring 
of the year show less than 50 per cent of Nosema-infected bees are 
likely to recover from the infection without treatment, showing that 
under such circumstances the infection is not transmitted within the 
hive, to any great extent at least. The fact that a colony may contain 
a small percentage of Nosema-infected bees throughout the year 
and not become heavily infected at any time furnishes further evi- 
dence that Nosema infection does not always spread with rapidity 
within the hive. It has been found that colonies becoming heavily 
infected through experimental inoculation in June, July, or August, 
are practically free from the infection within six weeks from the date 
of inoculation, showing again that the infection is not always readily 
transmitted within the hive. 



48 BULLETIN 780, IT. S. DEPARTMENT OF AGRICULTURE. 

Colonies may die out, or they may only become weakened by the 
disease. Each of these conditions invites robbing, which in a certain 
number of cases probably results in the transmission of the disease. 
The likelihood of the transmission of the disease through robbing, 
however, seems to be not nearly as great as in the case of the foul- 
broods. 

Uninoculated colonies in the experimental apiary have always 
remained practically free from infection, although colonies heavily 
infected as the result of experimental inoculations were present. 
This fact suggests that very little infection, if any, results either 
from the visit of healthy bees to flowers previously visited by infected 
ones, or, furthermore, from the straying or drifting of bees from 
infected to healthy colonies. 

The possibility that the queen may be infected and that infection 
will be transmitted by her to the other bees of the colony need give the 
apiarist no uneasiness, and no concern need be felt that drones will 
spread the disease in the apiary. 

Fear that Nosema-infection might be transmitted by hives which 
have housed infected colonies need not be entertained; neither is it to 
be feared that the hands or clothing of the beekeepers, or the tools used 
about an apiary, will serve as means for the transmission of the 
disease. Furthermore, the spread of the disease is not to be attrib- 
uted directly to winds. 

Theoretically it would seem that combs from Nosema-diseased 
colonies, if inserted into a healthy colony, would be the means of 
transmitting the disease and that the danger would extend over a 
period of a few weeks or months (p. 39). Experimentally it is 
shown, however, that such combs can be inserted immediately without 
transmitting the disorder, at least appreciably (p. 43). 

Evidence is yet to be obtained to prove that insects other than 
honeybees are susceptible to infection with Nosema apis. A few 
experiments made in which silkworms, maggots, and ants were 
inoculated with this parasite gave negative results. At the present 
time, therefore, there is no cause for fear that Nosema-disease will be 
transmitted as the result of a similar infection in other insects. 

DIAGNOSIS OF NOSEMA-DISEASE. . 

Nosema-disease usually can be diagnosed from the colony symptoms 
present together with the gross appearance of stomachs removed from 
adult bees of the colony. 

Weakness, especially in the spring of the year, should cause a sus- 
picion that the disease is present. The suspicion is strengthened if in 
such a colony the brood in general is normal, if the adult bees are not 
noticeably different in outward appearance or behavior from bees of 
healthy colonies, if the queen is present and if stores are abundant. 



NOSEMA-DISEASE. 49 

While the colony symptoms may justify a very strong suspicion that 
the disease is present, an examination of the stomachs from adult bees 
of the colony is necessary in making a definite diagnosis. The selec- 
tion of the proper sample for examination is important. In choosing 
samples it is advisable to take such bees as are most likely to show a 
high percentage Nosema-infected. Young workers, old shiny ones, 
and drones are, therefore, to be avoided. Workers from the field are 
naturally to be preferred. As bees carrying pollen are most readily 
recognized as being field bees, these are the ones usually sought. 
Sometimes it is more convenient to take bees carrying honey or water. 
Next to the field bees, preference should be had for bees from among 
those about the entrance of the hive. During the colder seasons of 
the year it is often necessary to take the samples from the brood- 
combs. 

Ten bees from a colony constitute a satisfactory sample as a rule. 
Ordinarily these are taken at the entrance with forceps. They 
are killed by pinching the thorax. All of the bees of the sample 
should be examined. 

In removing the stomach for examination the bee is held by the 
thorax between the thumb and index finger of one hand and with a 
pair of forceps held in the other the tip of the abdomen is seized and 
pulled gently. By this method the organs of the alimentary tract 
(PL I) forward to and including the stomach are easily obtained. 
Occasionally the proventriculus and honey sac are also removed by 
this procedure. The stomach is the most prominent of the organs 
removed and the one that is most readily recognized. 

If the stomach upon removal appears swollen and lighter in color 
than a healthy one, Nosema infection may be suspected; if it is chalk- 
white and easily torn, infection is very probable; should the tissues 
of the organ when crushed be milky in appearance, infection is 
practically certain. Usually the gross examination is sufficient for 
a definite diagnosis of the disease as encountered in nature. Some- 
times it is desirable however, to have such a diagnosis confirmed by a 
microscopic examination of the crushed tissues of the stomach. This 
is often the case in experimental studies. 

If infection is present in a bee the oval glistening spores of the 
parasite (fig. 4) usually will be found in very large numbers upon a 
microscopic examination of the crushed tissues of the stomach. No 
staining is needed. Addition of water to the mount is not necessary 
but it improves the preparation, permitting the spores to be seen more 
distinctly. Stomachs which become dry, after their removal and be- 
fore the examination is made, can be used readily by the addition of 
water. 



50 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 

Very few objects are encountered in the microscopic examination 
of the stomachs that are likely to be mistaken for the spores of 
Nosema apis. Occasionally yeasts are encountered. They occur, 
however, in small numbers only, as a rule; a variation in size is 
usually to be observed; and if stained they take the stain readily 
and intensely. The writer occasionally has encountered small oval 
bodies resembling spores which escape from pollen grains. They are 
found in comparatively small numbers when encountered, however, 
and are smaller than Nosema spores. What these bodies are has 
not been determined. 

In examining bees that have been dead of Nosema-disease for some 
time a portion of the contents of the abdomen is suspended in water 
on a slide and examined microscopically. The highly refractive oval 
spores of the parasite will be found if the bee was Nosema infected 
at the time of its death. Younger stages of the parasite will not be 
encountered under these conditions. 

Stages of the parasite that precede that of the spores may be 
recognized at times from fresh preparations. Forms approaching 
spores in appearance, which have been referred to as young spores, 
together with growing or vegetative forms appearing frequently as 
though they were in pairs (PL III, I), are seen occasionally. These 
younger forms are not likely to be recognized in preparations except 
in those made from bees recently killed and then only in small num- 
bers. They should not be depended upon in the making of the 
diagnosis. 

To determine very early stages of infection with Nosema apis the 
stomach of the suspected bee must be fixed, sectioned, and stained 
by laboratory methods. 1 The parasite is then found in the epithe- 
lial cells of the organ. 

Nosema-disease, like sacbrood, is quite prevalent among bees, and 
like sacbrood a small amount of infection may be present in a colony 
without producing any appreciable loss. When a diagnosis of the 
disease is being made in practical apiculture, therefore, considerable 
caution should be observed. A colony showing only a small per- 
centage of Nosema-inf ected bees and no other evidence of the disease 
is practically healthy. In reporting the presence of infection it 
would seem well to indicate in some way the amount of infection 
present. The percentage of infected bees among those examined 
might be given. 

*As a fixing fluid one containing a strong solution of mercuric chlorid can be recommended in studies 
on Nosemaapis. Heidenhain's iron hematoxylin is a verysatisfactorystainformuchofthework. Other 
fixers, especially those containing picric acid or formalin, have been used successfully. The sporesof 
Nosema apis are not readily stained by all stains. Pyfonin sometimes gives good results with methyl 
green as a counterstain. Alcoholic eosin applied for a considerable period, with methyl blue as acounter- 
stain, used on fixed smears made from fresh tissues, often results in desirable preparations. 



NOSEMA-DISEASE. 51 

In expressing a positive diagnosis the degree of infection could be 
indicated, for the present at least, by the terms "slight," "moder- 
ate," "heavy," and "very heavy." Slight infection by this scheme 
would indicate that not more than 10 per cent of the bees are infected 
and that no noticeable loss is to be anticipated from the infection; 
moderate infection would indicate that from 10 to 35 per cent are 
infected, that the colony will probably sustain losses from the dis- 
ease, but that the chances are good for recovery; heavy infection 
would indicate that from 30 to 60 per cent are infected, that the 
colony will most likely show weakness as a result of the disease, and 
that it may or may not die; and very heavy infection would indi- 
cate that more than 60 per cent are infected and that the colony 
will probably die as a result of the disease. 

While a definite diagnosis in regard to Nosema infection can 
always be made by laboratory methods (McCray and White, 1918), 
beekeepers in most instances can diagnose the disease sufficiently 
well for practical purposes in the apiary. Weakness should cause 
suspicion. If there is no other obvious cause for the weak condition 
a strengthened suspicion is justified. If, upon the removal of the 
stomachs of a few field bees (at least 10 should be examined), some 
white stomachs are found among them, the presence of Nosema- 
disease is quite certain. Should there still exist a doubt the organ 
should be examined further. If the tissues seem to tear easily and 
when crushed present a milky appearance, 1 it may be concluded that 
the colony is Nosema infected. 

DIFFERENTIAL DIAGNOSIS. 

Dysentery, paralysis, palsy, spring dwindling, Isle of Wight dis- 
ease, May pest, May sickness, abdominal distension, dry dysentery, 
dropsy, and disappearing trick are some of the many names which 
have been applied to disorders among adult bees. The disorders for 
which the names have been used have not been sufficiently well 
defined in all instances, however, to insure their positive diagnosis. 
From the facts at hand it seems probable that the number of adult 
diseases is small and that each disease, therefore, from time to time 
has had more than one name applied to it. It seems equally prob- 
able that some of the names used have been applied to more than 
one disease. 

Although little of a definite character is known concerning the 
disorders of adult bees in general, Nosema-disease is such a definite 
condition that its differentiation from other disorders should not be 
difficult. It is the only adult disease that can be diagnosed posi- 
tively at the present time by laboratory methods. 

* In testing the "milky appearance," crush the suspected stomach between two plates of clear glass. 



52 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 

DY8ENTERY. 

The term " dysentery" as applied to a disorder among adult bees 
is found in early beekeeping literature and is still encountered fre- 
quently. The spotting of the hive which is so often referred to as a 
symptom of dysenterj^and the absence of Nosema apis will' serve to 
distinguish it from Nosema-disease. 

PARALYSIS. 1 

The term "paralysis" has been widely used to designate a disease 
of adult bees. In this country the name usually is applied to a con- 
dition in which a large number of the bees of the affected colony die 
suddenly with the result that often a large mass of them is found 
in front of the hive. When this disorder is encountered usually only 
a colony here and there in the apiary is affected. Whether or not 
the disorder is infectious has not yet been determined. Time has 
permitted the making of only a few preliminary experiments on this 
disorder by the writer. The few which have been made and the 
facts as observed by practical beekeepers indicate that if the disease 
is infectious it is only slightly so. It is not likely, therefore, to 
spread to any great extent in the apiary. It can be differentiated 
from Nosema-disease by the absence of Nosema apis in the bees that 
have died of the disorder, and in the bees remaining in the colony. 

SPRING DWINDLING. 

It is very probable that more than one disorder has been referred 
to by the term "spring dwindling." When Nosema-disease was 
encountered by the beekeepers in the past, most likely it was often 
designated spring dwindling. Other conditions which are called 
spring dwindling may be differentiated from Nosema-disease by the 
fact that Nosema -apis is present in Nosema-disease and is absent in 
other conditions unless, of course, a mixed infection is present. 

ISLE OF WIGHT DISEASE. 

There has been encountered in many parts of England a disorder 
among adult bees from which heavy losses have been reported. The 
condition was described in 1906 by the beekeepers on the Isle of 
Wight, where apiaries had suffered heavy losses. 

Bullamore and Maiden (1912), of England, after studying the 
symptoms of the disease, arrived at the conclusion "that no one 
symptom is characteristic of the Isle of Wight disease, the only 
essential feature being the death of large numbers of bees within or 

* On account of the shaking or trembling movements sometimes manifested by individual bees affected, 
the term "palsy" has been used to designate the condition. As this term describes more accurately a 
marked symptom observed in the individual bee affected, it would seem to be a more appropriate one 
than "paralysis." 



NOSEMA-DISEASE. 53 

without the hive." They believed that the condition had been 
endemic in parts of England for many years, and shared with 
Graham-Smith the belief that a large amount of the losses among 
adult bees ascribed to it is due to Nosema infection. 

From the facts at hand it is not possible to state whether the Isle 
of Wight disease and Nosema-disease are one and the same disorder. 
Studies made on the Isle of Wight disease by English workers will 
most likely result in revealing further valuable facts concerning it 
(Anderson and Rennie, 1916). The writer examined one sample of 
adult bees from England taken from a colony suffering from Isle of 
Wight disease. No spores of Nosema apis were found in the sample. 
The results of the examination naturally prove nothing regarding 
the disease. 

For the present the American beekeeper should bear in mind that 
when Nosema-disease is given as the diagnosis, a condition having 
the destructiveness described for the Isle of Wight disease is not 
meant. 

OTHER DISEASES OP ADULT BEES. 

It is quite probable that other diseases of adult bees than those 
referred to here exist. If so, they have not yet been sufficiently 
studied to make their recognition possible, at least by laboratory 
methods. Such disorders could be differentiated from Nosema- 
disease by the absence in them of Nosema apis. As Nosema infection 
is very widely distributed among bees, the fact must always be 
borne in mind that Nosema infection may occur in a colony together 
with other bee diseases and be of secondary importance. This 
caution should never be overlooked. 

PROGNOSIS IN NOSEMA-DISEASE. 

The prognosis in Nosema-disease varies markedly and is dependent 
upon the conditions present. Of these conditions the percentage of 
Nosema-infected bees in the colony, the strength of the colony, the 
season of the year, and the environment of the apiary are among the 
more important factors which determine the outcome of the disease. 

The percentage of Nosema-infected bees in the colony may be very 
small, much less than 1 per cent, or it may be very large, reaching 
practically 100 per cent. Between these limits all degrees of infec- 
tion are encountered, the prognosis in each instance being different. 

As a rule colonies which in the spring of the year show less than 
10 per cent of Nosema-infected bees gain in strength and the losses 
are not detected. This is often true also in cases where the infection 
is somewhat greater than 10 per cent. When the number of infected 
bees approaches 50 per cent the colonies become noticeably weakened 
and in many instances death takes place. When more than 50 per 



54 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 

cent are infected they become weakened and usually die as a result 
of the infection. Generally speaking, therefore, it may be said that 
when a colony contains less than 10 per cent of Nosema-infected bees 
the prognosis is excellent; that when it contains more than 10 and less 
than 50 per cent the prognosis is fair; that when it contains more 
than 50 per cent the prognosis is unfavorable; and that when the 
number of Nosema-infected bees present approaches 100 per cent the 
prognosis is especially grave. 

In arriving at a decision as to the probable course and outcome of 
the infection the strength of the colony must also be considered. 
This factor, indeed, may be the deciding one. As a rule, the stronger 
the colony, the more favorable is the prognosis. 

In early spring heavy losses among the workers are not replaced 
and the colony weakens. During the active brood-rearing season, 
on the other hand, the bees dying of the infection are replaced by 
young bees. These young bees being free from infection and the 
transmission of the disease within the hive during summer being 
slight as a rule, the prognosis at this season of the year is favorable. 

Experimentally it is found that a single inoculation early in the 
spring will cause a colony to die as a result of the infection produced ; 
if inoculated somewhat later, however, the colony will weaken 
appreciably but will recover from the infection ; if inoculated during 
the active brood-rearing season the weakening effect resulting from 
the infection may not be appreciable ; if inoculated toward the close 
of the brood-rearing season the weakness resulting will be noticeable, 
but the colony may winter; and if inoculated later in the autumn 
or during the winter the colony will die as a result of the infection. 
It will be seen, therefore, that the prognosis in Nosema-disease in 
every case is dependent in some measure upon the season of the 
year, being more favorable in the active brood-rearing season than in 
any other. Indeed the season may play a major role in determining 
the course and outcome of the disease. 

The immediate environment of the apiary may possibly play a 
r61e in determining the prognosis. Opportunity for reinfection from 
without tends to vary the course and outcome of the disease. In this 
connection the nature of the water supply should not be overlooked. 

The extent to which the different races of bees vary in their suscep- 
tibility to the disease, the extent to which individual col®nies vary in 
their susceptibility, and the extent to which different strains of 
Nosema apis vary as to their virulence are not at all definitely known 
at the present time. The facts, however, indicate that in no instance 
is the variation particularly great. Much care should be exercised, 
therefore, in ascribing variations in losses from the disease to the two 
phenomena virulence of the germ and resistance of the host. 



NOSEMA-DISEASE. 55 

Whether a bee once infected ever recovers from the infection has 
not yet been established definitely. From what is known of diseases 
in man and animals one might expect recovery in a certain percent- 
age of Nosema-infected bees. The data at hand indicate that 
occasionally recovery does take place in the worker bee. This is 
suggested by the fact that among the last few workers alive in a 
colony, following a heavy infection resulting from an experimental 
inoculation, some have been found upon examination to be only 
slightly infected and still others to be free from infection. The 
only conclusion that can be drawn at the present time on this point 
is that if recovery from the infection ever takes place in the worker 
bee the cases are comparatively rare. 

Whether the prognosis is as grave in the case of an infected queen 
is not known. The facts at hand suggest that it probably is not. 
In the writer's experience less than 50 per cent of the queens in 
experimental colonies were found to be infected (Table I). Whether 
they had been infected and had recovered was not determined. 
The queens from colonies which had been inoculated from one to 
three weeks were found to be free from infection, indicating that 
infection was infrequent, at least within the period that workers 
and drones show the greatest percentage of infection. 

Death from Nosema infection does not take place for some 
time after infection. The length of time an infected worker lives 
depends in a large measure upon the season of the year. During the 
active bee season death takes place as a rule in less than one month 
but in more than two weeks. During winter the disease may run 
a course of two or three months or even more. Infected drones 
die sooner than infected workers, whereas infected queens probably 
live longer. This relation is to be expected since in healthy bees 
a somewhat similar relation exists. It is quite likely that the age 
of the bee when infected is not a negligible factor in determining 
the course of the disease. 

Finally it should be emphasized that the prognosis of Nosema 
infection, as it occurs in the United States, is not nearly so unfavor- 
able as has been reported for the Isle of Wight disease in England 
and for Nosema infection in Bavaria, Germany. It is, however, 
very similar to that of the infection as it has been reported from 
Australia (Price, 1910; Laidlow, 1911; and Beuhne, 1916). 



56 BULLETIN" 780, U. S. DEPARTMENT OF AGRICULTURE. 

SUMMARY AND CONCLUSIONS. 

The following statements concerning Nosema-disease seem to 
be justified from the facts recorded in the present paper: 

(1) Nosema-disease is an infectious disorder of adult bees caused 
by Nosema apis. 

(2) The disease is not particularly malignant in character, being 
in this respect more like sacbrood than the foulbroods. 

(3) Adult workers, drones, and queens are susceptible to infec- 
tion, but the brood is not. 

(4) The infecting agent Nosema apis is a protozoan that attacks 
the walls of the stomach and occasionally those of the Malpighian 
tubules. 

(5) A colony can be inoculated by feeding it sirup containing 
the crushed stomachs of infected bees. 

(6) One-tenth of the germs present in a single stomach are sufficient 
to produce marked infection in a colony. 

(7) Within a week following the inoculation the parasite can be 
found within the walls of the stomach. 

(8) Before the close of the second week infection can be determined 
by the gross appearance of the organ. 

(9) The disease can be produced at any season of the year by feed- 
ing inoculations. 

(10) Infected bees may be found at all seasons of the year, the 
highest percentage of infection occurring in the spring. 

(11) Nosema infection among bees occurs at least in Australia, 
Switzerland, Germany, Denmark, England, Canada, and the United 
States. This distribution shows that the occurrence of the disease 
is not dependent altogether upon climatic conditions. 

(12) The course of the disease is not affected directly by the 
character or quantity of food obtained and used by the bees. 

(13) A sluggish body of water, if near an apiary and used by bees 
as a water supply, and the robbing of diseased colonies, must be 
considered for the present as two probable sources of infection. 

(14) The transmission of the disease through the medium of flowers 
is not to be feared. 

(15) The hands and clothing of the apiarist, the tools used about 
an apiary, and winds need ilot be feared as means by which the 
disease is spread. 

(16) Hives which have housed infected colonies need not be dis- 
infected and combs from such colonies are not a likely means for the 
transmission of the disease. 

(17) Bees dead of the disease about the apiary are not likely to 
cause infection unless they serve to contaminate the water supply. 

(18) Nosema apis suspended in water is destroyed by heating for 10 
minutes at about 136° F. (58° C). 



STOSEMA-DISEASE. 57 

(19) Suspended in honey, Nosema apis is destroyed by heating at 
about 138° F. (59° C.). 

(20) Nosema apis, drying at room and outdoor temperatures, re- 
spectively, remained virulent for about 2 months, at incubator tem- 
perature about 3 weeks, and in a refrigerator about 7 J months. 

(21) Nosema apis was destroyed in the presence of fermentative 
processes in a 20 per cent honey solution in 3 days at incubator tem- 
perature and in 9 days at outdoor temperature. In a 10 per cent 
sugar solution it was destroyed in from 7 to 11 days at room tem- 
perature. 

(22) Nosema apis resisted putrefactive processes for 5 days at 
incubator temperature, for 2 weeks at room temperature, and for more 
than 3 weeks at outdoor temperature. 

(23) Nosema apis when dry was destroyed in from 15 to 32 hours 
by direct exposure to the sun's rays. 

(24) Nosema apis suspended in water was destroyed by exposure 
to the sun's rays in from 37 to 51 hours. 

(25) Nosema apis if suspended in honey and exposed to the sun's 
rays frequently will be destroyed on account of the temperature of 
the honey which results from the exposure. 

(26) Nosema apis remained virulent in honey for from 2 to 4 
months at room temperature. 

(27) Nosema apis in the bodies of dead bees ceased to be virulent 
in one week at incubator temperature, in 4 weeks at room tempera- 
ture, in 6 weeks at outdoor temperature, and in 4 months in a refrig- 
erator. 

(28) Nosema apis in the bodies of dead bees lying on the soil ceased 
to be virulent in from 44 to 71 days. 

(29) Nosema apis is readily destroyed by carbolic acid, a 1 per cent 
aqueous solution destroying it in less than 10 minutes. 

(30) The time element which by the experiments is shown to be 
sufficient for the destruction of Nosema apis should be increased 
somewhat to insure their destruction in practical apiculture. 

(31) The prognosis in Nosema-disease varies markedly from excel- 
lent, in case of strong colonies with a comparatively small percentage 
of Nosema-infected bees, to very grave, in case of weak ones with a 
high percentage of infected bees. 

(32) From a technical point of view the results here given must be 
considered as being approximate only. They are, however, in most 
instances sufficient for practical purposes. 



58 BULLETIN 780, U. S. DEPARTMENT OF AGRICULTURE. 

LITERATURE CITED. 

Since 1909 numerous articles relating to Nosema-disease have 
appeared in the bee journals. Among these are to be found reviews 
of papers detailing the results of investigations which have been made 
on this disorder of bees. The following list of papers, together with 
the bibliographies contained in them, furnishes a fairly complete 
reference to the literature on this disease. 

Angst, H. 

1913. Die Nosemakrankheit der Bienen. In Schweizerische Bienen-Zeitung, 
Aarau, n. f. Jahrg. 36, No. 3, p. 97-104, March. 
Anderson, John, and Rennie, John. 

1916. Observations and experiments bearing on "Isle of Wight" disease in hive 
bees. In Proc. Roy. Phys. Soc. Edinb., Session 1915-1916, v. 20, pt. 1, 
p. 23-61, 1 pi. 
Bahr, L. 

1915. Sygdomme hos Honningbien og dens Yngel. Meddelelser fra den Kgl. 

Veterinaer.-og Landboh^jskoles Serumlaboratorium, XXXVII, 109 p., 

11 %. 

Literature, p. 108-109. 

1916. Die Krankheiten der Honigbiene und ihrer Brut. Hannover. 19 p. 
Sonder-Abdruck aus Nr. 28 u. 29 der Deutschen Tierarztlichen Wochen- 

schrift (24 Jahrg. 1916). (Mitteilungen aus dem Serum-Laboratorium 
der Koniglichen Danischen Veterinar. und Landwirtshaftlichen Hoch- 
schule. 
Beuhne, F. R. 

1911. Dysentery in bees and Nosema apis. In Jour. Dept. Agr. Victoria, 

Australia, v. 9, pt. 8, p. 550-551, August 10. 
1913. Diseases of bees, continued. In Jour. Dept. Agr. Victoria, Australia, 

v. 11, pt. 8, p. 487-493, 4 figs., August. 
1916. Nosema apis in Victoria. In Jour. Dept. Agr. Victoria, Australia, v. 14, 
pt. 10, p. 629-632, October. 
Brotbeck. 

1857. Der Fadenpilz als Bienenkrankheit. In Bienen-zeitung, v. 13, no. 18, 
p. 215, September 10. 
Burri, R. 

1912. Tatigkeitsbericht der S(&weiz-milchwirtschaftlichen Anstalt-Bern-Liebe- 

feld pro 1911 erstattet an das schweiz Landwirtschaf tsdepartement. In 
Landwirtschaftliches Jahrbuch der Schweiz, Jahrg. 26, p. 469-491. 
Page 471. Im apistischen Betrieb. 
Donhoff and Leuckart. 

1857. Ueber den Fadenpilz im Darm der Biene (mit einer lithographirten 
Beilage). In Bienen-zeitung, [Eichstadt], Jahrg. 13, Nr. 6, p. 66-67, 
72, March 30. 
1857. Ueber die Ansteckungsfahigkeit. In Bienen-zeitung, [Eichstadt], Jahrg. 

13, Nr. 16 and 17, p. 199, August 27. 
1857. Ueber den Verbreitung der Pilzsucht. In Bienen-Zeitung, [Eichstadt], 
Jahrg. 13, Nr. 18, p. 210, September 10. 
Fantham, A. B., and Porter, Annie. 

1911. A bee-disease due to a protozoal parasite (Nosema apis). In Proc. Zool. 
Soc. London, 1911, pt. Ill, p. 625-626, September. 



NOSEMA-DISEASE. 59 

Graham-Smith, G. S., Fantham, H. B., Porter, Annie, Bullamore, G. W., and 
Malden, W. 

1912. Report on the Isle of Wight bee disease (Microsporidiosis). In Supple- 

ment no. 8 to the Jour. Bd. Agr. [London], v. 19, no. 2, 143 p., 5 pi., 
May. 

Bibliography, p. 139-143. 

1913. Further report on the Isle of Wight bee disease (Microsporidiosis). In 

Supplement no. 10 to the Jour. Bd. Agr. [London], v. 20, no. 4, 47 p., 
July 

Bibliography, p. 46-47. 

Laidlow, W. 

1911. Bee diseases investigation. In Australasian beekeeper, v. 13, no. 2, p. 25, 
August 15. 
Maassen and Nithack. 

1910. Uber die Ruhr der Bienen, In Mitteilungen aus der Kaiserlichen biolo- 

gischen Anstalt fur Land- und Forstwirtschaft, Heft. 10, p. 39-42, March. 
Maassen, A. 

1911. Zur Aetiologie und Epidemiologie der Ruhr bei den Bienen volkern. In 

Mitteilungen aus der Kaiserlichen biologischen Anstalt fiir Land- und 

Forstwirtschaft, Heft 11, p. 50- 54, March. 
McCray, A. H., and White, G. F. 

1918. The diagnosis of bee diseases by laboratory methods. In U. S. Dept. Agr. 

Bui. 671, 15 p., 2 pi., June 21. 
McIndoo, N. E. 

1916. The sense organs on the mouth parts of the honey bee. In Smithsonian 

Miscellaneous Collections, v. 65, no. 14, 55 p., 10 figs., Jan. 12. [Publi- 
cation 2381.] 
Nussbaumer, Thos. 

1912. Einige Erfahrungen iiber die Nosemakrankheit. In Schweizerische' 

Bienenzeitung, n. f. Jahrg. 35 (whole ser. 48), no. 1, p. 30-33, January. 
Price, G. A. E. 

1910. Bee mortality in the Stawell District. In Jour. Dept. Agr. Victoria, 
Australia, v. 8, pt. 1, p. 58-62, [2] fig., January 10. 
Snodgrass, R. E. 

1910. The anatomy of the honey bee. U. S. Dept. Agr. Bur. Ent. Tech. Ser. 18, 

162 p., 57 fig., May 28. 
Bibliography, p. 148-150. 

Stempell, W. 

, 1909. Ueber Nosema bombycis Nageli nebst Bemerkungen iiber Mikrophoto- 
graphie mit gewohnlichem und ultra violettem Licht. In Arch. f. Pro- 
tistenkunde, Jena, v. 16, no. 3, p. 281-358, 1 fig., pi. 19-25. 
White, G. F. 

1914. Destruction of germs of infectious bee diseases by heating. TJ. S. Dept. 

Agr. Bui. 92, 8 p., May 15. 

1917. Sacbrood. U. S. Dept. Agr. Bui. 431, 55 p., 4 pi., 33 fig. 

1918. A note on the muscular coat of the ventriculus of the honey bee (Apis 

mellifica). JnProc. Ent. Soc. Wash., v. 20, no. 7, p. 152-154, December 4. 

Zander, Enoch. 

1909. Tierische Parasiten als Krankheitserreger bei der Biene. In Leipziger 
Bienenzeitung, Jahrg. 24, Heft 10, p. 147-150, figs., Oct., and Heft 11, 
p. 164-166, Nov. (Also in Miinchener Bienenzeitung, 1909, Heft 9.) 

Zander, Enoch. 

1911. Die Krankheiten und Schadlinge der erwachsenen Bienen. Stuttgart, 

42 p., 8 pi. (Handbuch der Bienenkunde II.) 



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